Polypeptides having catalase activity and polynucleotides encoding same

ABSTRACT

Provided are isolated polypeptides having catalase activity and polynucleotides encoding the polypeptides. Also provided are nucleic acid constructs, vectors and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a 35 U.S.C. 371 national application ofPCT/CN2012/086946 filed Dec. 19, 2012 which claims priority or thebenefit under 35 U.S.C. 119 of Chinese PCT application no.PCT/CN2011/084230 filed Dec. 19, 2011 and U.S. provisional applicationNo. 61/582,913 filed Jan. 4, 2012, the contents of which are fullyincorporated herein by reference.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

This invention was made with Government support under CooperativeAgreement DE-FC36-08GO18080 awarded by the Department of Energy. Thegovernment has certain rights in this invention.

REFERENCE TO A SEQUENCE LISTING

This application contains a Sequence Listing in computer readable form,which is incorporated herein by reference.

BACKGROUND OF THE INVENTION

Field of the Invention

The present invention relates to polypeptides having catalase activityand catalytic domains, and polynucleotides encoding the polypeptides,and catalytic domains. The invention also relates to nucleic acidconstructs, vectors, and host cells comprising the polynucleotides aswell as methods of producing and using the polypeptides, and catalyticdomains.

Description of the Related Art

Catalases [hydrogen peroxide:hydrogen peroxide oxidoreductases (EC1.11.1.6)] are enzymes which catalyze the conversion of hydrogenperoxide (H₂O₂) to oxygen (O₂) and water (H₂O). These ubiquitous enzymeshave been purified from a variety of animal tissues, plants andmicroorganisms (Chance and Maehly, 1955, Methods Enzymol. 2: 764-791).

Catalase preparations are used commercially for diagnostic enzyme kits,for the enzymatic production of sodium gluconate from glucose, for theneutralization of H₂O₂ waste, and for the removal of H₂O₂ and/orgeneration of O₂ in foods and beverages.

WO 92/17571 discloses a catalase, which retain activity at highertemperature and pH than other known catalases, from strains ofScytalidium and Humicola. UNIPROT:AIDJU9 discloses a deduced amino acidsequence of catalase from Neosartorya fischeri. UNIPROT:P30266 disclosesa catalase from Bacillus pseudofirmu. UNIPROT:P42234 discloses acatalase polypeptide from Bacillus subtilis. JP2007143405-A discloses acatalase from Thermoascus aurantiacus.

The present invention provides polypeptides having catalase activity andpolynucleotides encoding the polypeptides.

SUMMARY OF THE INVENTION

The present invention relates to isolated polypeptides having catalaseactivity selected from the group consisting of:

(a) a polypeptide having at least 83% sequence identity to the maturepolypeptide of SEQ ID NO: 8, at least at least 76% sequence identity tothe mature polypeptide of SEQ ID NO: 2, at least 60% sequence identityto the mature polypeptide of SEQ ID NO: 4, or at least 60% sequenceidentity to the mature polypeptide of SEQ ID NO: 6;

(b) a polypeptide encoded by a polynucleotide that hybridizes under low,medium, medium-high, high, or very high stringency conditions with (i)the mature polypeptide coding sequence of SEQ ID NO: 7, the maturepolypeptide coding sequence of SEQ ID NO: 1, the mature polypeptidecoding sequence of SEQ ID NO: 3, or the mature polypeptide codingsequence of SEQ ID NO: 5, (ii) the cDNA sequence thereof, or (iii) thefull-length complement of (i) or (ii);

(c) a polypeptide encoded by a polynucleotide having at least 83%sequence identity to the mature polypeptide coding sequence of SEQ IDNO: 7, at least 76% sequence identity to the mature polypeptide codingsequence of SEQ ID NO: 1, at least 60% sequence identity to the maturepolypeptide coding sequence of SEQ ID NO: 3, or at least 60% sequenceidentity to the mature polypeptide coding sequence of SEQ ID NO: 5; orthe cDNA sequence thereof;

(d) a variant of the mature polypeptide of SEQ ID NO: 8, a variant ofthe mature polypeptide of SEQ ID NO: 2, a variant of the maturepolypeptide of SEQ ID NO: 4, or a variant of the mature polypeptide ofSEQ ID NO: 6, comprising a substitution, deletion, and/or insertion atone or more (e.g., several) positions; and

(e) a fragment of the polypeptide of (a), (b), (c), or (d) that hascatalase activity.

The present invention also relates to isolated polypeptides comprising acatalytic domain selected from the group consisting of:

(a) a catalytic domain having at least 83% sequence identity to aminoacids 20 to 740 of SEQ ID NO: 8, a catalytic domain having at least 76%sequence identity to amino acids 17 to 723 of SEQ ID NO: 2, a catalyticdomain having at least 60% sequence identity to amino acids 38 to 723 ofSEQ ID NO: 4, or a catalytic domain having at least 60% sequenceidentity to amino acids 38 to 711 of SEQ ID NO: 6;

(b) a polypeptide encoded by a polynucleotide that hybridizes under low,medium, medium-high, high, or very high stringency conditions with (i)nucleotides 58 to 2473 of SEQ ID NO: 7, nucleotides 49 to 2601 of SEQ IDNO: 1, nucleotides 112 to 2687 of SEQ ID NO: 3, or nucleotides 112 to2652 of SEQ ID NO: 5, (ii) the cDNA sequence thereof, or (iii) thefull-length complement of (i) or (ii);

(c) a catalytic domain encoded by a polynucleotide having at least 83%sequence identity to nucleotides 58 to 2473 of SEQ ID NO: 7, a catalyticdomain encoded by a polynucleotide having at least 76% sequence identityto nucleotides 49 to 2601 of SEQ ID NO: 1, a catalytic domain encoded bya polynucleotide having at least 60% sequence identity to nucleotides112 to 2687 of SEQ ID NO: 3, or a catalytic domain encoded by apolynucleotide having at least 60% sequence identity to nucleotides 112to 2652 of SEQ ID NO: 5;

(d) a variant of amino acids 20 to 740 of SEQ ID NO: 8, a variant ofamino acids 17 to 723 of SEQ ID NO: 2, a variant of amino acids 38 to723 of SEQ ID NO: 4, or a variant of amino acids 38 to 711 of SEQ ID NO:6, comprising a substitution, deletion, and/or insertion at one or more(e.g., several) positions; and

(e) a fragment of the catalytic domain of (a), (b), (c), or (d) whichhas catalase activity.

The present invention also relates to isolated polynucleotides encodingthe polypeptides of the present invention; nucleic acid constructs;recombinant expression vectors; recombinant host cells comprising thepolynucleotides; and methods of producing the polypeptides.

The present invention also relates to processes for degrading orconverting a cellulosic material, comprising: treating the cellulosicmaterial with an enzyme composition in the presence of a polypeptidehaving catalase activity of the present invention. In one aspect, theprocesses further comprise recovering the degraded or convertedcellulosic material.

The present invention also relates to processes of producing afermentation product, comprising: (a) saccharifying a cellulosicmaterial with an enzyme composition in the presence of a polypeptidehaving catalase activity of the present invention; (b) fermenting thesaccharified cellulosic material with one or more (e.g., several)fermenting microorganisms to produce the fermentation product; and (c)recovering the fermentation product from the fermentation.

The present invention also relates to processes of fermenting acellulosic material, comprising: fermenting the cellulosic material withone or more (e.g., several) fermenting microorganisms, wherein thecellulosic material is saccharified with an enzyme composition in thepresence of a polypeptide having catalase activity of the presentinvention. In one aspect, the fermenting of the cellulosic materialproduces a fermentation product. In another aspect, the processesfurther comprise recovering the fermentation product from thefermentation.

The present invention also relates to a polynucleotide encoding a signalpeptide comprising or consisting of amino acids 1 to 19 of SEQ ID NO: 8,amino acids 1 to 16 of SEQ ID NO: 2, amino acids 1 to 20 of SEQ ID NO:4, or amino acids 1 to 24 of SEQ ID NO: 6, which is operably linked to agene encoding a protein; nucleic acid constructs, expression vectors,and recombinant host cells comprising the polynucleotides; and methodsof producing a protein.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the genomic DNA sequence (SEQ ID NO: 1) and the deducedamino acid sequence (SEQ ID NO: 2) of a Malbranchea cinnamomea catalasegene.

FIG. 2 shows the genomic DNA sequence (SEQ ID NO: 3) and the deducedamino acid sequence (SEQ ID NO: 4) of a Rhizomucor pusillus catalasegene.

FIG. 3 shows the genomic DNA sequence (SEQ ID NO: 5) and the deducedamino acid sequence (SEQ ID NO: 6) of a Rhizomucor pusillus catalasegene.

FIG. 4 shows the genomic DNA sequence (SEQ ID NO: 7) and the deducedamino acid sequence (SEQ ID NO: 8) of a Penicillium emersonii catalasegene.

FIG. 5 shows a restriction map of pCat_ZY582303_121.

FIG. 6 shows a restriction map of pCat_ZY654893_6661.

FIG. 7 shows a restriction map of pCat_ZY654878_5541.

FIG. 8 shows a restriction map of pCat_PE04230007241.

DEFINITIONS

Catalase: The term “catalase activity” is defined herein as ahydrogen-peroxide:hydrogen-peroxide oxidoreductase activity (EC1.11.1.6) that catalyzes the conversion of 2H₂O₂ to O₂+2 H₂O. Forpurposes of the present invention, catalase activity is determinedaccording to U.S. Pat. No. 5,646,025. One unit of catalase activityequals the amount of enzyme that catalyzes the oxidation of 1 μmole ofhydrogen peroxide under the assay conditions. Alternatively, thecatalase activity can be determined using the procedure described inExamples 17 and 18 of the present invention.

In one aspect, the polypeptides of the present invention have at least20%, e.g., at least 40%, at least 50%, at least 60%, at least 70%, atleast 80%, at least 90%, at least 95%, or at least 100% of the catalaseactivity of the mature polypeptide of SEQ ID NO: 2, the maturepolypeptide of SEQ ID NO: 4, the mature polypeptide of SEQ ID NO: 6, orthe mature polypeptide of SEQ ID NO: 8.

Acetylxylan esterase: The term “acetylxylan esterase” means acarboxylesterase (EC 3.1.1.72) that catalyzes the hydrolysis of acetylgroups from polymeric xylan, acetylated xylose, acetylated glucose,alpha-napthyl acetate, and p-nitrophenyl acetate. For purposes of thepresent invention, acetylxylan esterase activity is determined using 0.5mM p-nitrophenylacetate as substrate in 50 mM sodium acetate pH 5.0containing 0.01% TWEEN™ 20 (polyoxyethylene sorbitan monolaurate). Oneunit of acetylxylan esterase is defined as the amount of enzyme capableof releasing 1 μmole of p-nitrophenolate anion per minute at pH 5, 25°C.

Allelic variant: The term “allelic variant” means any of two or morealternative forms of a gene occupying the same chromosomal locus.Allelic variation arises naturally through mutation, and may result inpolymorphism within populations. Gene mutations can be silent (no changein the encoded polypeptide) or may encode polypeptides having alteredamino acid sequences. An allelic variant of a polypeptide is apolypeptide encoded by an allelic variant of a gene.

Alpha-L-arabinofuranosidase: The term “alpha-L-arabinofuranosidase”means an alpha-L-arabinofuranoside arabinofuranohydrolase (EC 3.2.1.55)that catalyzes the hydrolysis of terminal non-reducingalpha-L-arabinofuranoside residues in alpha-L-arabinosides. The enzymeacts on alpha-L-arabinofuranosides, alpha-L-arabinans containing (1,3)-and/or (1,5)-linkages, arabinoxylans, and arabinogalactans.Alpha-L-arabinofuranosidase is also known as arabinosidase,alpha-arabinosidase, alpha-L-arabinosidase, alpha-arabinofuranosidase,polysaccharide alpha-L-arabinofuranosidase, alpha-L-arabinofuranosidehydrolase, L-arabinosidase, or alpha-L-arabinanase. For purposes of thepresent invention, alpha-L-arabinofuranosidase activity is determinedusing 5 mg of medium viscosity wheat arabinoxylan (MegazymeInternational Ireland, Ltd., Bray, Co. Wicklow, Ireland) per ml of 100mM sodium acetate pH 5 in a total volume of 200 μl for 30 minutes at 40°C. followed by arabinose analysis by AMINEX® HPX-87H columnchromatography (Bio-Rad Laboratories, Inc., Hercules, Calif., USA).

Alpha-glucuronidase: The term “alpha-glucuronidase” means analpha-D-glucosiduronate glucuronohydrolase (EC 3.2.1.139) that catalyzesthe hydrolysis of an alpha-D-glucuronoside to D-glucuronate and analcohol. For purposes of the present invention, alpha-glucuronidaseactivity is determined according to de Vries, 1998, J. Bacteriol. 180:243-249. One unit of alpha-glucuronidase equals the amount of enzymecapable of releasing 1 μmole of glucuronic or 4-O-methylglucuronic acidper minute at pH 5, 40° C.

Beta-glucosidase: The term “beta-glucosidase” means a beta-D-glucosideglucohydrolase (E.C. 3.2.1.21) that catalyzes the hydrolysis of terminalnon-reducing beta-D-glucose residues with the release of beta-D-glucose.For purposes of the present invention, beta-glucosidase activity isdetermined using p-nitrophenyl-beta-D-glucopyranoside as substrateaccording to the procedure of Venturi et al., 2002, Extracellularbeta-D-glucosidase from Chaetomium thermophilum var. coprophilum:production, purification and some biochemical properties, J. BasicMicrobiol. 42: 55-66. One unit of beta-glucosidase is defined as 1.0μmole of p-nitrophenolate anion produced per minute at 25° C., pH 4.8from 1 mM p-nitrophenyl-beta-D-glucopyranoside as substrate in 50 mMsodium citrate containing 0.01% TWEEN® 20.

Beta-xylosidase: The term “beta-xylosidase” means a beta-D-xylosidexylohydrolase (E.C. 3.2.1.37) that catalyzes the exo-hydrolysis of shortbeta (1→4)-xylooligosaccharides to remove successive D-xylose residuesfrom non-reducing termini. For purposes of the present invention, oneunit of beta-xylosidase is defined as 1.0 μmole of p-nitrophenolateanion produced per minute at 40° C., pH 5 from 1 mMp-nitrophenyl-beta-D-xyloside as substrate in 100 mM sodium citratecontaining 0.01% TWEEN® 20.

Catalytic domain: The term “catalytic domain” means the region of anenzyme containing the catalytic machinery of the enzyme.

cDNA: The term “cDNA” means a DNA molecule that can be prepared byreverse transcription from a mature, spliced, mRNA molecule obtainedfrom a eukaryotic or prokaryotic cell. cDNA lacks intron sequences thatmay be present in the corresponding genomic DNA. The initial, primaryRNA transcript is a precursor to mRNA that is processed through a seriesof steps, including splicing, before appearing as mature spliced mRNA.

Cellobiohydrolase: The term “cellobiohydrolase” means a1,4-beta-D-glucan cellobiohydrolase (E.C. 3.2.1.91 and E.C. 3.2.1.176)that catalyzes the hydrolysis of 1,4-beta-D-glucosidic linkages incellulose, cellooligosaccharides, or any beta-1,4-linked glucosecontaining polymer, releasing cellobiose from the reducing end(cellobiohydrolase I) or non-reducing end (cellobiohydrolase II) of thechain (Teeri, 1997, Crystalline cellulose degradation: New insight intothe function of cellobiohydrolases, Trends in Biotechnology 15: 160-167;Teeri et al., 1998, Trichoderma reesei cellobiohydrolases: why soefficient on crystalline cellulose?, Biochem. Soc. Trans. 26: 173-178).Cellobiohydrolase activity is determined according to the proceduresdescribed by Lever et al., 1972, Anal. Biochem. 47: 273-279; vanTilbeurgh et al., 1982, FEBS Letters, 149: 152-156; van Tilbeurgh andClaeyssens, 1985, FEBS Letters, 187: 283-288; and Tomme et al., 1988,Eur. J. Biochem. 170: 575-581. In the present invention, the Tomme etal. method can be used to determine cellobiohydrolase activity.

Cellulolytic enzyme or cellulase: The term “cellulolytic enzyme” or“cellulase” means one or more (e.g., several) enzymes that hydrolyze acellulosic material. Such enzymes include endoglucanase(s),cellobiohydrolase(s), beta-glucosidase(s), or combinations thereof. Thetwo basic approaches for measuring cellulolytic activity include: (1)measuring the total cellulolytic activity, and (2) measuring theindividual cellulolytic activities (endoglucanases, cellobiohydrolases,and beta-glucosidases) as reviewed in Zhang et al., Outlook forcellulase improvement: Screening and selection strategies, 2006,Biotechnology Advances 24: 452-481. Total cellulolytic activity isusually measured using insoluble substrates, including Whatman No1filter paper, microcrystalline cellulose, bacterial cellulose, algalcellulose, cotton, pretreated lignocellulose, etc. The most common totalcellulolytic activity assay is the filter paper assay using Whatman No1filter paper as the substrate. The assay was established by theInternational Union of Pure and Applied Chemistry (IUPAC) (Ghose, 1987,Measurement of cellulase activities, Pure Appl. Chem. 59: 257-68).

For purposes of the present invention, cellulolytic enzyme activity isdetermined by measuring the increase in hydrolysis of a cellulosicmaterial by cellulolytic enzyme(s) under the following conditions: 1-50mg of cellulolytic enzyme protein/g of cellulose in PCS (or otherpretreated cellulosic material) for 3-7 days at a suitable temperature,e.g., 50° C., 55° C., or 60° C., compared to a control hydrolysiswithout addition of cellulolytic enzyme protein. Typical conditions are1 ml reactions, washed or unwashed PCS, 5% insoluble solids, 50 mMsodium acetate pH 5, 1 mM MnSO₄, 50° C., 55° C., or 60° C., 72 hours,sugar analysis by AMINEX® HPX-87H column (Bio-Rad Laboratories, Inc.,Hercules, Calif., USA).

Cellulosic material: The term “cellulosic material” means any materialcontaining cellulose. The predominant polysaccharide in the primary cellwall of biomass is cellulose, the second most abundant is hemicellulose,and the third is pectin. The secondary cell wall, produced after thecell has stopped growing, also contains polysaccharides and isstrengthened by polymeric lignin covalently cross-linked tohemicellulose. Cellulose is a homopolymer of anhydrocellobiose and thusa linear beta-(1-4)-D-glucan, while hemicelluloses include a variety ofcompounds, such as xylans, xyloglucans, arabinoxylans, and mannans incomplex branched structures with a spectrum of substituents. Althoughgenerally polymorphous, cellulose is found in plant tissue primarily asan insoluble crystalline matrix of parallel glucan chains.Hemicelluloses usually hydrogen bond to cellulose, as well as to otherhemicelluloses, which help stabilize the cell wall matrix.

Cellulose is generally found, for example, in the stems, leaves, hulls,husks, and cobs of plants or leaves, branches, and wood of trees. Thecellulosic material can be, but is not limited to, agricultural residue,herbaceous material (including energy crops), municipal solid waste,pulp and paper mill residue, waste paper, and wood (including forestryresidue) (see, for example, Wiselogel et al., 1995, in Handbook onBioethanol (Charles E. Wyman, editor), pp. 105-118, Taylor & Francis,Wash. D.C.; Wyman, 1994, Bioresource Technology 50: 3-16; Lynd, 1990,Applied Biochemistry and Biotechnology 24/25: 695-719; Mosier et al,1999, Recent Progress in Bioconversion of Lignocellulosics, in Advancesin Biochemical Engineering/Biotechnology, T. Scheper, managing editor,Volume 65, pp. 23-40, Springer-Verlag, New York). It is understoodherein that the cellulose may be in the form of lignocellulose, a plantcell wall material containing lignin, cellulose, and hemicellulose in amixed matrix. In a preferred aspect, the cellulosic material is anybiomass material. In another preferred aspect, the cellulosic materialis lignocellulose, which comprises cellulose, hemicelluloses, andlignin.

In one aspect, the cellulosic material is agricultural residue. Inanother aspect, the cellulosic material is herbaceous material(including energy crops). In another aspect, the cellulosic material ismunicipal solid waste. In another aspect, the cellulosic material ispulp and paper mill residue. In another aspect, the cellulosic materialis waste paper. In another aspect, the cellulosic material is wood(including forestry residue).

In another aspect, the cellulosic material is arundo. In another aspect,the cellulosic material is bagasse. In another aspect, the cellulosicmaterial is bamboo. In another aspect, the cellulosic material is corncob. In another aspect, the cellulosic material is corn fiber. Inanother aspect, the cellulosic material is corn stover. In anotheraspect, the cellulosic material is miscanthus. In another aspect, thecellulosic material is orange peel. In another aspect, the cellulosicmaterial is rice straw. In another aspect, the cellulosic material isswitchgrass. In another aspect, the cellulosic material is wheat straw.

In another aspect, the cellulosic material is aspen. In another aspect,the cellulosic material is eucalyptus. In another aspect, the cellulosicmaterial is fir. In another aspect, the cellulosic material is pine. Inanother aspect, the cellulosic material is poplar. In another aspect,the cellulosic material is spruce. In another aspect, the cellulosicmaterial is willow.

In another aspect, the cellulosic material is algal cellulose. Inanother aspect, the cellulosic material is bacterial cellulose. Inanother aspect, the cellulosic material is cotton linter. In anotheraspect, the cellulosic material is filter paper. In another aspect, thecellulosic material is microcrystalline cellulose. In another aspect,the cellulosic material is phosphoric-acid treated cellulose.

In another aspect, the cellulosic material is an aquatic biomass. Asused herein the term “aquatic biomass” means biomass produced in anaquatic environment by a photosynthesis process. The aquatic biomass canbe algae, emergent plants, floating-leaf plants, or submerged plants.

The cellulosic material may be used as is or may be subjected topretreatment, using conventional methods known in the art, as describedherein. In a preferred aspect, the cellulosic material is pretreated.

Cellulolytic enzyme or cellulase: The term “cellulolytic enzyme” or“cellulase” means one or more (e.g., several) enzymes that hydrolyze acellulosic material. Such enzymes include endoglucanase(s),cellobiohydrolase(s), beta-glucosidase(s), or combinations thereof. Thetwo basic approaches for measuring cellulolytic activity include: (1)measuring the total cellulolytic activity, and (2) measuring theindividual cellulolytic activities (endoglucanases, cellobiohydrolases,and beta-glucosidases) as reviewed in Zhang et al., Outlook forcellulase improvement: Screening and selection strategies, 2006,Biotechnology Advances 24: 452-481. Total cellulolytic activity isusually measured using insoluble substrates, including Whatman No1filter paper, microcrystalline cellulose, bacterial cellulose, algalcellulose, cotton, pretreated lignocellulose, etc. The most common totalcellulolytic activity assay is the filter paper assay using Whatman No1filter paper as the substrate. The assay was established by theInternational Union of Pure and Applied Chemistry (IUPAC) (Ghose, 1987,Measurement of cellulase activities, Pure Appl. Chem. 59: 257-68).

For purposes of the present invention, cellulolytic enzyme activity isdetermined by measuring the increase in hydrolysis of a cellulosicmaterial by cellulolytic enzyme(s) under the following conditions: 1-50mg of cellulolytic enzyme protein/g of cellulose in PCS (or otherpretreated cellulosic material) for 3-7 days at a suitable temperature,e.g., 50° C., 55° C., or 60° C., compared to a control hydrolysiswithout addition of cellulolytic enzyme protein. Typical conditions are1 ml reactions, washed or unwashed PCS, 5% insoluble solids, 50 mMsodium acetate pH 5, 1 mM MnSO₄, 50° C., 55° C., or 60° C., 72 hours,sugar analysis by AMINEX® HPX-87H column (Bio-Rad Laboratories, Inc.,Hercules, Calif., USA).

Coding sequence: The term “coding sequence” means a polynucleotide,which directly specifies the amino acid sequence of a polypeptide. Theboundaries of the coding sequence are generally determined by an openreading frame, which begins with a start codon such as ATG, GTG, or TTGand ends with a stop codon such as TAA, TAG, or TGA. The coding sequencemay be a genomic DNA, cDNA, synthetic DNA, or a combination thereof.

Control sequences: The term “control sequences” means nucleic acidsequences necessary for expression of a polynucleotide encoding a maturepolypeptide of the present invention. Each control sequence may benative (i.e., from the same gene) or foreign (i.e., from a differentgene) to the polynucleotide encoding the polypeptide or native orforeign to each other. Such control sequences include, but are notlimited to, a leader, polyadenylation sequence, propeptide sequence,promoter, signal peptide sequence, and transcription terminator. At aminimum, the control sequences include a promoter, and transcriptionaland translational stop signals. The control sequences may be providedwith linkers for the purpose of introducing specific restriction sitesfacilitating ligation of the control sequences with the coding region ofthe polynucleotide encoding a polypeptide.

Endoglucanase: The term “endoglucanase” means anendo-1,4-(1,3;1,4)-beta-D-glucan 4-glucanohydrolase (E.C. 3.2.1.4) thatcatalyzes endohydrolysis of 1,4-beta-D-glycosidic linkages in cellulose,cellulose derivatives (such as carboxymethyl cellulose and hydroxyethylcellulose), lichenin, beta-1,4 bonds in mixed beta-1,3 glucans such ascereal beta-D-glucans or xyloglucans, and other plant materialcontaining cellulosic components. Endoglucanase activity can bedetermined by measuring reduction in substrate viscosity or increase inreducing ends determined by a reducing sugar assay (Zhang et al., 2006,Biotechnology Advances 24: 452-481). For purposes of the presentinvention, endoglucanase activity is determined using carboxymethylcellulose (CMC) as substrate according to the procedure of Ghose, 1987,Pure and Appl. Chem. 59: 257-268, at pH 5, 40° C.

Expression: The term “expression” includes any step involved in theproduction of a polypeptide including, but not limited to,transcription, post-transcriptional modification, translation,post-translational modification, and secretion.

Expression vector: The term “expression vector” means a linear orcircular DNA molecule that comprises a polynucleotide encoding apolypeptide and is operably linked to control sequences that provide forits expression.

Family 61 glycoside hydrolase: The term “Family 61 glycoside hydrolase”or “Family GH61” or “GH61” means a polypeptide falling into theglycoside hydrolase Family 61 according to Henrissat B., 1991, Aclassification of glycosyl hydrolases based on amino-acid sequencesimilarities, Biochem. J. 280: 309-316, and Henrissat B., and BairochA., 1996, Updating the sequence-based classification of glycosylhydrolases, Biochem. J. 316: 695-696. The enzymes in this family wereoriginally classified as a glycoside hydrolase family based onmeasurement of very weak endo-1,4-beta-D-glucanase activity in onefamily member. The structure and mode of action of these enzymes arenon-canonical and they cannot be considered as bona fide glycosidases.However, they are kept in the CAZy classification on the basis of theircapacity to enhance the breakdown of lignocellulose when used inconjunction with a cellulase or a mixture of cellulases.

Feruloyl esterase: The term “feruloyl esterase” means a4-hydroxy-3-methoxycinnamoyl-sugar hydrolase (EC 3.1.1.73) thatcatalyzes the hydrolysis of 4-hydroxy-3-methoxycinnamoyl (feruloyl)groups from esterified sugar, which is usually arabinose in “natural”substrates, to produce ferulate (4-hydroxy-3-methoxycinnamate). Feruloylesterase is also known as ferulic acid esterase, hydroxycinnamoylesterase, FAE-III, cinnamoyl ester hydrolase, FAEA, cinnAE, FAE-I, orFAE-II. For purposes of the present invention, feruloyl esteraseactivity is determined using 0.5 mM p-nitrophenylferulate as substratein 50 mM sodium acetate pH 5.0. One unit of feruloyl esterase equals theamount of enzyme capable of releasing 1 μmole of p-nitrophenolate anionper minute at pH 5, 25° C.

Fragment: The term “fragment” means a polypeptide having one or more(e.g., several) amino acids absent from the amino and/or carboxylterminus of a mature polypeptide main; wherein the fragment has catalaseactivity. In another aspect, a fragment contains at least 614 amino acidresidues, e.g., at least 650 amino acid residues or at least 686 aminoacid residues of SEQ ID NO: 8. In one aspect, a fragment contains atleast 604 amino acid residues, e.g., at least 640 amino acid residues orat least 676 amino acid residues of SEQ ID NO: 2. In another aspect, afragment contains at least 602 amino acid residues, e.g., at least 638amino acid residues or at least 674 amino acid residues of SEQ ID NO: 4.In another aspect, a fragment contains at least 588 amino acid residues,e.g., at least 623 amino acid residues or at least 658 amino acidresidues of SEQ ID NO: 6.

Hemicellulolytic enzyme or hemicellulase: The term “hemicellulolyticenzyme” or “hemicellulase” means one or more (e.g., several) enzymesthat hydrolyze a hemicellulosic material. See, for example, Shallom, D.and Shoham, Y. Microbial hemicellulases. Current Opinion InMicrobiology, 2003, 6(3): 219-228). Hemicellulases are key components inthe degradation of plant biomass. Examples of hemicellulases include,but are not limited to, an acetylmannan esterase, an acetylxylanesterase, an arabinanase, an arabinofuranosidase, a coumaric acidesterase, a feruloyl esterase, a galactosidase, a glucuronidase, aglucuronoyl esterase, a mannanase, a mannosidase, a xylanase, and axylosidase. The substrates of these enzymes, the hemicelluloses, are aheterogeneous group of branched and linear polysaccharides that arebound via hydrogen bonds to the cellulose microfibrils in the plant cellwall, crosslinking them into a robust network. Hemicelluloses are alsocovalently attached to lignin, forming together with cellulose a highlycomplex structure. The variable structure and organization ofhemicelluloses require the concerted action of many enzymes for itscomplete degradation. The catalytic modules of hemicellulases are eitherglycoside hydrolases (GHs) that hydrolyze glycosidic bonds, orcarbohydrate esterases (CEs), which hydrolyze ester linkages of acetateor ferulic acid side groups. These catalytic modules, based on homologyof their primary sequence, can be assigned into GH and CE families. Somefamilies, with an overall similar fold, can be further grouped intoclans, marked alphabetically (e.g., GH-A). A most informative andupdated classification of these and other carbohydrate active enzymes isavailable in the Carbohydrate-Active Enzymes (CAZy) database.Hemicellulolytic enzyme activities can be measured according to Ghoseand Bisaria, 1987, Pure & Appl. Chem. 59: 1739-1752, at a suitabletemperature, e.g., 50° C., 55° C., or 60° C., and pH, e.g., 5.0 or 5.5.

High stringency conditions: The term “high stringency conditions” meansfor probes of at least 100 nucleotides in length, prehybridization andhybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/ml shearedand denatured salmon sperm DNA, and 50% formamide, following standardSouthern blotting procedures for 12 to 24 hours. The carrier material isfinally washed three times each for 15 minutes using 2×SSC, 0.2% SDS at65° C.

Host cell: The term “host cell” means any cell type that is susceptibleto transformation, transfection, transduction, or the like with anucleic acid construct or expression vector comprising a polynucleotideof the present invention. The term “host cell” encompasses any progenyof a parent cell that is not identical to the parent cell due tomutations that occur during replication.

Isolated: The term “isolated” means a substance in a form or environmentthat does not occur in nature. Non-limiting examples of isolatedsubstances include (1) any non-naturally occurring substance, (2) anysubstance including, but not limited to, any enzyme, variant, nucleicacid, protein, peptide or cofactor, that is at least partially removedfrom one or more or all of the naturally occurring constituents withwhich it is associated in nature; (3) any substance modified by the handof man relative to that substance found in nature; or (4) any substancemodified by increasing the amount of the substance relative to othercomponents with which it is naturally associated (e.g., recombinantproduction in a host cell; multiple copies of a gene encoding thesubstance; and use of a stronger promoter than the promoter naturallyassociated with the gene encoding the substance)

Low stringency conditions: The term “low stringency conditions” meansfor probes of at least 100 nucleotides in length, prehybridization andhybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/ml shearedand denatured salmon sperm DNA, and 25% formamide, following standardSouthern blotting procedures for 12 to 24 hours. The carrier material isfinally washed three times each for 15 minutes using 2×SSC, 0.2% SDS at50° C.

Mature polypeptide: The term “mature polypeptide” means a polypeptide inits final form following translation and any post-translationalmodifications, such as N-terminal processing, C-terminal truncation,glycosylation, phosphorylation, etc. In another aspect, the maturepolypeptide is amino acids 20 to 741 of SEQ ID NO: 8 based on theSignalP program that predicts amino acids 1 to 19 of SEQ ID NO: 8 are asignal peptide. In one aspect, the mature polypeptide is amino acids 17to 729 of SEQ ID NO: 2 based on the SignalP program (Nielsen et al.,1997, Protein Engineering 10: 1-6) that predicts amino acids 1 to 16 ofSEQ ID NO: 2 are a signal peptide. In another aspect, the maturepolypeptide is amino acids 21 to 730 of SEQ ID NO: 4 based on theSignalP program that predicts amino acids 1 to 20 of SEQ ID NO: 4 are asignal peptide. In another aspect, the mature polypeptide is amino acids25 to 717 of SEQ ID NO: 6 based on the SignalP program that predictsamino acids 1 to 24 of SEQ ID NO: 6 are a signal peptide. It is known inthe art that a host cell may produce a mixture of two of more differentmature polypeptides (i.e., with a different C-terminal and/or N-terminalamino acid) expressed by the same polynucleotide. It is also known inthe art that different host cells process polypeptides differently, andthus, one host cell expressing a polynucleotide may produce a differentmature polypeptide (e.g., having a different C-terminal and/orN-terminal amino acid) as compared to another host cell expressing thesame polynucleotide.

Mature polypeptide coding sequence: The term “mature polypeptide codingsequence” means a polynucleotide that encodes a mature polypeptidehaving catalase activity. In another aspect, the mature polypeptidecoding sequence is nucleotides 58 to 2476 of SEQ ID NO: 7 or the cDNAsequence thereof based on the SignalP program that predicts nucleotides1 to 57 of SEQ ID NO: 7 encode a signal peptide. In one aspect, themature polypeptide coding sequence is nucleotides 49 to 2619 of SEQ IDNO: 1 or the cDNA sequence thereof based on the SignalP program (Nielsenet al., 1997, supra) that predicts nucleotides 1 to 48 of SEQ ID NO: 1encode a signal peptide. In another aspect, the mature polypeptidecoding sequence is nucleotides 61 to 2708 of SEQ ID NO: 3 or the cDNAsequence thereof based on the SignalP program that predicts nucleotides1 to 60 of SEQ ID NO: 3 encode a signal peptide. In another aspect, themature polypeptide coding sequence is nucleotides 73 to 2670 of SEQ IDNO: 5 or the cDNA sequence thereof based on the SignalP program thatpredicts nucleotides 1 to 72 of SEQ ID NO: 5 encode a signal peptide.

Medium stringency conditions: The term “medium stringency conditions”means for probes of at least 100 nucleotides in length, prehybridizationand hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/mlsheared and denatured salmon sperm DNA, and 35% formamide, followingstandard Southern blotting procedures for 12 to 24 hours. The carriermaterial is finally washed three times each for 15 minutes using 2×SSC,0.2% SDS at 55° C.

Medium-high stringency conditions: The term “medium-high stringencyconditions” means for probes of at least 100 nucleotides in length,prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200micrograms/ml sheared and denatured salmon sperm DNA, and either 35%formamide, following standard Southern blotting procedures for 12 to 24hours. The carrier material is finally washed three times each for 15minutes using 2×SSC, 0.2% SDS at 60° C.

Nucleic acid construct: The term “nucleic acid construct” means anucleic acid molecule, either single- or double-stranded, which isisolated from a naturally occurring gene or is modified to containsegments of nucleic acids in a manner that would not otherwise exist innature or which is synthetic, which comprises one or more controlsequences.

Operably linked: The term “operably linked” means a configuration inwhich a control sequence is placed at an appropriate position relativeto the coding sequence of a polynucleotide such that the controlsequence directs expression of the coding sequence.

Polypeptide having cellulolytic enhancing activity: The term“polypeptide having cellulolytic enhancing activity” means a GH61polypeptide that catalyzes the enhancement of the hydrolysis of acellulosic material by enzyme having cellulolytic activity. For purposesof the present invention, cellulolytic enhancing activity is determinedby measuring the increase in reducing sugars or the increase of thetotal of cellobiose and glucose from the hydrolysis of a cellulosicmaterial by cellulolytic enzyme under the following conditions: 1-50 mgof total protein/g of cellulose in pretreated corn stover (PCS), whereintotal protein is comprised of 50-99.5% w/w cellulolytic enzyme proteinand 0.5-50% w/w protein of a GH61 polypeptide having cellulolyticenhancing activity for 1-7 days at a suitable temperature, e.g., 50° C.,55° C., or 60° C., and a suitable pH such 4-9, e.g., 5.0 or 5.5,compared to a control hydrolysis with equal total protein loadingwithout cellulolytic enhancing activity (1-50 mg of cellulolyticprotein/g of cellulose in PCS). In a preferred aspect, a mixture ofCELLUCLAST® 1.5 L (Novozymes A/S, Bagsværd, Denmark) in the presence of2-3% of total protein weight Aspergillus oryzae beta-glucosidase(recombinantly produced in Aspergillus oryzae according to WO 02/095014)or 2-3% of total protein weight Aspergillus fumigatus beta-glucosidase(recombinantly produced in Aspergillus oryzae as described in WO2002/095014) of cellulase protein loading is used as the source of thecellulolytic activity.

The GH61 polypeptides having cellulolytic enhancing activity enhance thehydrolysis of a cellulosic material catalyzed by enzyme havingcellulolytic activity by reducing the amount of cellulolytic enzymerequired to reach the same degree of hydrolysis preferably at least1.01-fold, e.g., at least 1.05-fold, at least 1.10-fold, at least1.25-fold, at least 1.5-fold, at least 2-fold, at least 3-fold, at least4-fold, at least 5-fold, at least 10-fold, or at least 20-fold.

Pretreated corn stover: The term “PCS” or “Pretreated Corn Stover” meansa cellulosic material derived from corn stover by treatment with heatand dilute sulfuric acid, alkaline pretreatment, or neutralpretreatment.

Sequence identity: The relatedness between two amino acid sequences orbetween two nucleotide sequences is described by the parameter “sequenceidentity”.

For purposes of the present invention, the sequence identity between twoamino acid sequences is determined using the Needleman-Wunsch algorithm(Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implementedin the Needle program of the EMBOSS package (EMBOSS: The EuropeanMolecular Biology Open Software Suite, Rice et al., 2000, Trends Genet.16: 276-277), preferably version 3.0.0, 5.0.0 or later. The parametersused are gap open penalty of 10, gap extension penalty of 0.5, and theEBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The outputof Needle labeled “longest identity” (obtained using the -nobriefoption) is used as the percent identity and is calculated as follows:(Identical Residues×100)/(Length of Alignment−Total Number of Gaps inAlignment)

For purposes of the present invention, the sequence identity between twodeoxyribonucleotide sequences is determined using the Needleman-Wunschalgorithm (Needleman and Wunsch, 1970, supra) as implemented in theNeedle program of the EMBOSS package (EMBOSS: The European MolecularBiology Open Software Suite, Rice et al., 2000, supra), preferablyversion 3.0.0, 5.0.0 or later. The parameters used are gap open penaltyof 10, gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version ofNCBI NUC4.4) substitution matrix. The output of Needle labeled “longestidentity” (obtained using the -nobrief option) is used as the percentidentity and is calculated as follows:(Identical Deoxyribonucleotides×100)/(Length of Alignment−Total Numberof Gaps in Alignment)

Subsequence: The term “subsequence” means a polynucleotide having one ormore (e.g., several) nucleotides absent from the 5′ and/or 3′ end of amature polypeptide coding sequence; wherein the subsequence encodes afragment having catalase activity. In another aspect, a subsequencecontains at least 1842 nucleotides, e.g., at least 1950 nucleotides orat least 2058 nucleotides of SEQ ID NO: 7. In one aspect, a subsequencecontains at least 1812 nucleotides, e.g., at least 1920 nucleotides orat least 2028 nucleotides of SEQ ID NO: 1. In another aspect, asubsequence contains at least 1806 nucleotides, e.g., at least 1914nucleotides or at least 2022 nucleotides of SEQ ID NO: 3. In anotheraspect, a subsequence contains at least 1764 nucleotides, e.g., at least1869 nucleotides or at least 1974 nucleotides of SEQ ID NO: 5.

Variant: The term “variant” means a polypeptide having catalase activitycomprising an alteration, i.e., a substitution, insertion, and/ordeletion, at one or more (e.g., several) positions. A substitution meansreplacement of the amino acid occupying a position with a differentamino acid; a deletion means removal of the amino acid occupying aposition; and an insertion means adding an amino acid adjacent to andimmediately following the amino acid occupying a position.

Very high stringency conditions: The term “very high stringencyconditions” means for probes of at least 100 nucleotides in length,prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200micrograms/ml sheared and denatured salmon sperm DNA, and 50% formamide,following standard Southern blotting procedures for 12 to 24 hours. Thecarrier material is finally washed three times each for 15 minutes using2×SSC, 0.2% SDS at 70° C.

Very low stringency conditions: The term “very low stringencyconditions” means for probes of at least 100 nucleotides in length,prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200micrograms/ml sheared and denatured salmon sperm DNA, and 25% formamide,following standard Southern blotting procedures for 12 to 24 hours. Thecarrier material is finally washed three times each for 15 minutes using2×SSC, 0.2% SDS at 45° C.

Xylan-containing material: The term “xylan-containing material” meansany material comprising a plant cell wall polysaccharide containing abackbone of beta-(1-4)-linked xylose residues. Xylans of terrestrialplants are heteropolymers possessing a beta-(1-4)-D-xylopyranosebackbone, which is branched by short carbohydrate chains. They compriseD-glucuronic acid or its 4-O-methyl ether, L-arabinose, and/or variousoligosaccharides, composed of D-xylose, L-arabinose, D- or L-galactose,and D-glucose. Xylan-type polysaccharides can be divided into homoxylansand heteroxylans, which include glucuronoxylans,(arabino)glucuronoxylans, (glucurono)arabinoxylans, arabinoxylans, andcomplex heteroxylans. See, for example, Ebringerova et al., 2005, Adv.Polym. Sci. 186: 1-67.

In the processes of the present invention, any material containing xylanmay be used. In a preferred aspect, the xylan-containing material islignocellulose.

Xylan degrading activity or xylanolytic activity: The term “xylandegrading activity” or “xylanolytic activity” means a biologicalactivity that hydrolyzes xylan-containing material. The two basicapproaches for measuring xylanolytic activity include: (1) measuring thetotal xylanolytic activity, and (2) measuring the individual xylanolyticactivities (e.g., endoxylanases, beta-xylosidases, arabinofuranosidases,alpha-glucuronidases, acetylxylan esterases, feruloyl esterases, andalpha-glucuronyl esterases). Recent progress in assays of xylanolyticenzymes was summarized in several publications including Biely andPuchard, 2006, Recent progress in the assays of xylanolytic enzymes,Journal of the Science of Food and Agriculture 86(11): 1636-1647;Spanikova and Biely, 2006, Glucuronoyl esterase—Novel carbohydrateesterase produced by Schizophyllum commune, FEBS Letters 580(19):4597-4601; Herrmann, Vrsanska, Jurickova, Hirsch, Biely, and Kubicek,1997, The beta-D-xylosidase of Trichoderma reesei is a multifunctionalbeta-D-xylan xylohydrolase, Biochemical Journal 321: 375-381.

Total xylan degrading activity can be measured by determining thereducing sugars formed from various types of xylan, including, forexample, oat spelt, beechwood, and larchwood xylans, or by photometricdetermination of dyed xylan fragments released from various covalentlydyed xylans. The most common total xylanolytic activity assay is basedon production of reducing sugars from polymeric 4-O-methylglucuronoxylan as described in Bailey, Biely, Poutanen, 1992,Interlaboratory testing of methods for assay of xylanase activity,Journal of Biotechnology 23(3): 257-270. Xylanase activity can also bedetermined with 0.2% AZCL-arabinoxylan as substrate in 0.01% TRITON®X-100 (4-(1,1,3,3-tetramethylbutyl)phenyl-polyethylene glycol) and 200mM sodium phosphate buffer pH 6 at 37° C. One unit of xylanase activityis defined as 1.0 μmole of azurine produced per minute at 37° C., pH 6from 0.2% AZCL-arabinoxylan as substrate in 200 mM sodium phosphate pH 6buffer.

For purposes of the present invention, xylan degrading activity isdetermined by measuring the increase in hydrolysis of birchwood xylan(Sigma Chemical Co., Inc., St. Louis, Mo., USA) by xylan-degradingenzyme(s) under the following typical conditions: 1 ml reactions, 5mg/ml substrate (total solids), 5 mg of xylanolytic protein/g ofsubstrate, 50 mM sodium acetate pH 5, 50° C., 24 hours, sugar analysisusing p-hydroxybenzoic acid hydrazide (PHBAH) assay as described byLever, 1972, A new reaction for colorimetric determination ofcarbohydrates, Anal. Biochem 47: 273-279.

Xylanase: The term “xylanase” means a 1,4-beta-D-xylan-xylohydrolase(E.C. 3.2.1.8) that catalyzes the endohydrolysis of 1,4-beta-D-xylosidiclinkages in xylans. For purposes of the present invention, xylanaseactivity is determined with 0.2% AZCL-arabinoxylan as substrate in 0.01%TRITON® X-100 and 200 mM sodium phosphate buffer pH 6 at 37° C. One unitof xylanase activity is defined as 1.0 μmole of azurine produced perminute at 37° C., pH 6 from 0.2% AZCL-arabinoxylan as substrate in 200mM sodium phosphate pH 6 buffer.

DETAILED DESCRIPTION OF THE INVENTION

Polypeptides Having Catalase Activity

In an embodiment, the present invention relates to isolated polypeptideshaving a sequence identity to the mature polypeptide of SEQ ID NO: 8 ofat least 83%, e.g., at least 84%, at least 85%, at least 86%, at least87%, at least 88%, at least 89%, at least 90%, at least 91%, at least92%, at least 93%, at least 94%, at least 95%, at least 96%, at least97%, at least 98%, at least 99%, or 100%, which have catalase activity.In an embodiment, the present invention relates to isolated polypeptideshaving a sequence identity to the mature polypeptide of SEQ ID NO: 2 ofat least 76%, e.g., at least 77%, at least 78%, at least 79%, at least80%, at least 81%, at least 82%, at least 83%, at least 84%, at least85%, at least 86%, at least 87%, at least 88%, at least 89%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%,which have catalase activity. In an embodiment, the present inventionrelates to isolated polypeptides having a sequence identity to themature polypeptide of SEQ ID NO: 4 of at least 60%, e.g., at least 65%,at least 70%, at least 75%, at least 78%, at least 80%, at least 81%, atleast 82%, at least 83%, at least 84%, at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100%, which have catalaseactivity. In an embodiment, the present invention relates to isolatedpolypeptides having a sequence identity to the mature polypeptide of SEQID NO: 6 of at least 60%, e.g., at least 65%, at least 70%, at least75%, at least 78%, at least 80%, at least 81%, at least 82%, at least83%, at least 84%, at least 85%, at least 86%, at least 87%, at least88%, at least 89%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, at least 99%, or 100%, which have catalase activity. In one aspect,the polypeptides differ by up to 10 amino acids, e.g., 1, 2, 3, 4, 5, 6,7, 8, 9 or 10, from the mature polypeptide of SEQ ID NO: 8, the maturepolypeptide of SEQ ID NO: 2, the mature polypeptide of SEQ ID NO: 4, orthe mature polypeptide of SEQ ID NO: 6.

A polypeptide of the present invention preferably comprises or consistsof the amino acid sequence of SEQ ID NO: 8, SEQ ID NO: 2, SEQ ID NO: 4,or SEQ ID NO: 6 or an allelic variant thereof; or is a fragment thereofhaving catalase activity. In another aspect, the polypeptide comprisesor consists of the mature polypeptide of SEQ ID NO: 8, the maturepolypeptide of SEQ ID NO: 2, the mature polypeptide of SEQ ID NO: 4, orthe mature polypeptide of SEQ ID NO: 6. In another aspect, thepolypeptide comprises or consists of amino acids 20 to 741 of SEQ ID NO:8, amino acids 17 to 729 of SEQ ID NO: 2, amino acids 21 to 730 of SEQID NO: 4, or amino acids 25 to 717 of SEQ ID NO: 6.

In another embodiment, the present invention relates to an isolatedpolypeptide having catalase activity encoded by a polynucleotide thathybridizes under very low stringency conditions, low stringencyconditions, medium stringency conditions, medium-high stringencyconditions, high stringency conditions, or very high stringencyconditions with (i) the mature polypeptide coding sequence of SEQ ID NO:7, the mature polypeptide coding sequence of SEQ ID NO: 1, the maturepolypeptide coding sequence of SEQ ID NO: 3, or the mature polypeptidecoding sequence of SEQ ID NO: 5, (ii) the cDNA sequence thereof, or(iii) the full-length complement of (i) or (ii) (Sambrook et al., 1989,Molecular Cloning, A Laboratory Manual, 2d edition, Cold Spring Harbor,N.Y.).

The polynucleotide of SEQ ID NO: 7, SEQ ID NO: 1, SEQ ID NO: 3, or SEQID NO: 5, or a subsequence thereof, as well as the polypeptide of SEQ IDNO: 8, SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6, or a fragmentthereof, may be used to design nucleic acid probes to identify and cloneDNA encoding polypeptides having catalase activity from strains ofdifferent genera or species according to methods well known in the art.In particular, such probes can be used for hybridization with thegenomic DNA or cDNA of a cell of interest, following standard Southernblotting procedures, in order to identify and isolate the correspondinggene therein. Such probes can be considerably shorter than the entiresequence, but should be at least 15, e.g., at least 25, at least 35, orat least 70 nucleotides in length. Preferably, the nucleic acid probe isat least 100 nucleotides in length, e.g., at least 200 nucleotides, atleast 300 nucleotides, at least 400 nucleotides, at least 500nucleotides, at least 600 nucleotides, at least 700 nucleotides, atleast 800 nucleotides, or at least 900 nucleotides in length. Both DNAand RNA probes can be used. The probes are typically labeled fordetecting the corresponding gene (for example, with ³²P, ³H, ³⁵S,biotin, or avidin). Such probes are encompassed by the presentinvention.

A genomic DNA or cDNA library prepared from such other strains may bescreened for DNA that hybridizes with the probes described above andencodes a polypeptide having catalase activity. Genomic or other DNAfrom such other strains may be separated by agarose or polyacrylamidegel electrophoresis, or other separation techniques. DNA from thelibraries or the separated DNA may be transferred to and immobilized onnitrocellulose or other suitable carrier material. In order to identifya clone or DNA that hybridizes with SEQ ID NO: 7, SEQ ID NO: 1, SEQ IDNO: 3, or SEQ ID NO: 5, or the mature polypeptide coding sequencethereof, or a subsequence thereof, the carrier material is used in aSouthern blot.

For purposes of the present invention, hybridization indicates that thepolynucleotides hybridize to a labeled nucleic acid probe correspondingto (i) SEQ ID NO: 7, SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5; (ii)the mature polypeptide coding sequence of SEQ ID NO: 7, the maturepolypeptide coding sequence of SEQ ID NO: 1, the mature polypeptidecoding sequence of SEQ ID NO: 3, or the mature polypeptide codingsequence of SEQ ID NO: 5; (iii) the cDNA sequence thereof; (iv) thefull-length complement thereof; or (v) a subsequence thereof; under verylow to very high stringency conditions. Molecules to which the nucleicacid probe hybridizes under these conditions can be detected using, forexample, X-ray film or any other detection means known in the art.

In one aspect, the nucleic acid probe is a polynucleotide that encodesthe polypeptide of SEQ ID NO: 8, SEQ ID NO: 2, SEQ ID NO: 4, or SEQ IDNO: 6, or the mature polypeptide thereof; or a fragment thereof. Inanother aspect, the nucleic acid probe is SEQ ID NO: 7, SEQ ID NO: 1,SEQ ID NO: 3, or SEQ ID NO: 5; or the cDNA sequence thereof. In oneaspect, the nucleic acid probe is the mature polypeptide coding sequenceof SEQ ID NO: 7, the mature polypeptide coding sequence of SEQ ID NO: 1,the mature polypeptide coding sequence of SEQ ID NO: 3, or the maturepolypeptide coding sequence of SEQ ID NO: 5.

In another embodiment, the present invention relates to isolatedpolypeptides having catalase activity encoded by a polynucleotide havinga sequence identity to the mature polypeptide coding sequence of SEQ IDNO: 7, or the cDNA sequence thereof, of at least 83%, e.g., at least84%, at least 85%, at least 86%, at least 87%, at least 88%, at least89%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, at least99%, or 100%. In another embodiment, the present invention relates toisolated polypeptides having catalase activity encoded by apolynucleotide having a sequence identity to the mature polypeptidecoding sequence of SEQ ID NO: 1, or the cDNA sequence thereof, of atleast 76%, e.g., at least 77%, at least 78%, at least 79%, at least 80%,at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, atleast 86%, at least 87%, at least 88%, at least 89%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100%. In anotherembodiment, the present invention relates to an isolated polypeptidehaving catalase activity encoded by a polynucleotide having a sequenceidentity to the mature polypeptide coding sequence of SEQ ID NO: 3, orthe cDNA sequence thereof, of at least 60%, e.g., at least 65%, at least70%, at least 75%, at least 78%, at least 80%, at least 81%, at least82%, at least 83%, at least 84%, at least 85%, at least 86%, at least87%, at least 88%, at least 89%, at least 90%, at least 91%, at least92%, at least 93%, at least 94%, at least 95%, at least 96%, at least97%, at least 98%, at least 99%, or 100%. In another embodiment, thepresent invention relates to an isolated polypeptide having catalaseactivity encoded by a polynucleotide having a sequence identity to themature polypeptide coding sequence of SEQ ID NO: 5, or the cDNA sequencethereof, of at least 60%, e.g., at least 65%, at least 70%, at least75%, at least 78%, at least 80%, at least 81%, at least 82%, at least83%, at least 84%, at least 85%, at least 86%, at least 87%, at least88%, at least 89%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, at least 99%, or 100%.

In another embodiment, the present invention relates to variants of themature polypeptide of SEQ ID NO: 8, variants of the mature polypeptideof SEQ ID NO: 2, variants of the mature polypeptide of SEQ ID NO: 4, orvariants of the mature polypeptide of SEQ ID NO: 6, comprising asubstitution, deletion, and/or insertion at one or more (e.g., several)positions. In an embodiment, the number of amino acid substitutions,deletions and/or insertions introduced into the mature polypeptide ofSEQ ID NO: 8, the mature polypeptide of SEQ ID NO: 2, the maturepolypeptide of SEQ ID NO: 4, or the mature polypeptide of SEQ ID NO: 6,is up to 10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. The amino acidchanges may be of a minor nature, that is conservative amino acidsubstitutions or insertions that do not significantly affect the foldingand/or activity of the protein; small deletions, typically of 1-30 aminoacids; small amino- or carboxyl-terminal extensions, such as anamino-terminal methionine residue; a small linker peptide of up to 20-25residues; or a small extension that facilitates purification by changingnet charge or another function, such as a poly-histidine tract, anantigenic epitope or a binding domain.

Examples of conservative substitutions are within the groups of basicamino acids (arginine, lysine and histidine), acidic amino acids(glutamic acid and aspartic acid), polar amino acids (glutamine andasparagine), hydrophobic amino acids (leucine, isoleucine and valine),aromatic amino acids (phenylalanine, tryptophan and tyrosine), and smallamino acids (glycine, alanine, serine, threonine and methionine). Aminoacid substitutions that do not generally alter specific activity areknown in the art and are described, for example, by H. Neurath and R. L.Hill, 1979, In, The Proteins, Academic Press, New York. Commonsubstitutions are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr,Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile,Leu/Val, Ala/Glu, and Asp/Gly.

Alternatively, the amino acid changes are of such a nature that thephysico-chemical properties of the polypeptides are altered. Forexample, amino acid changes may improve the thermal stability of thepolypeptide, alter the substrate specificity, change the pH optimum, andthe like.

Essential amino acids in a polypeptide can be identified according toprocedures known in the art, such as site-directed mutagenesis oralanine-scanning mutagenesis (Cunningham and Wells, 1989, Science 244:1081-1085). In the latter technique, single alanine mutations areintroduced at every residue in the molecule, and the resultant mutantmolecules are tested for catalase activity to identify amino acidresidues that are critical to the activity of the molecule. See also,Hilton et al., 1996, J. Biol. Chem. 271: 4699-4708. The active site ofthe enzyme or other biological interaction can also be determined byphysical analysis of structure, as determined by such techniques asnuclear magnetic resonance, crystallography, electron diffraction, orphotoaffinity labeling, in conjunction with mutation of putative contactsite amino acids. See, for example, de Vos et al., 1992, Science 255:306-312; Smith et al., 1992, J. Mol. Biol. 224: 899-904; Wlodaver etal., 1992, FEBS Lett. 309: 59-64. The identity of essential amino acidscan also be inferred from an alignment with a related polypeptide.

Single or multiple amino acid substitutions, deletions, and/orinsertions can be made and tested using known methods of mutagenesis,recombination, and/or shuffling, followed by a relevant screeningprocedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988,Science 241: 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA86: 2152-2156; WO 95/17413; or WO 95/22625. Other methods that can beused include error-prone PCR, phage display (e.g., Lowman et al., 1991,Biochemistry 30: 10832-10837; U.S. Pat. No. 5,223,409; WO 92/06204), andregion-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Neret al., 1988, DNA 7: 127).

Mutagenesis/shuffling methods can be combined with high-throughput,automated screening methods to detect activity of cloned, mutagenizedpolypeptides expressed by host cells (Ness et al., 1999, NatureBiotechnology 17: 893-896). Mutagenized DNA molecules that encode activepolypeptides can be recovered from the host cells and rapidly sequencedusing standard methods in the art. These methods allow the rapiddetermination of the importance of individual amino acid residues in apolypeptide.

The polypeptide may be a hybrid polypeptide in which a region of onepolypeptide is fused at the N-terminus or the C-terminus of a region ofanother polypeptide.

The polypeptide may be a fusion polypeptide or cleavable fusionpolypeptide in which another polypeptide is fused at the N-terminus orthe C-terminus of the polypeptide of the present invention. A fusionpolypeptide is produced by fusing a polynucleotide encoding anotherpolypeptide to a polynucleotide of the present invention. Techniques forproducing fusion polypeptides are known in the art, and include ligatingthe coding sequences encoding the polypeptides so that they are in frameand that expression of the fusion polypeptide is under control of thesame promoter(s) and terminator. Fusion polypeptides may also beconstructed using intein technology in which fusion polypeptides arecreated post-translationally (Cooper et al., 1993, EMBO J. 12:2575-2583; Dawson et al., 1994, Science 266: 776-779).

A fusion polypeptide can further comprise a cleavage site between thetwo polypeptides. Upon secretion of the fusion protein, the site iscleaved releasing the two polypeptides.

Examples of cleavage sites include, but are not limited to, the sitesdisclosed in Martin et al., 2003, J. Ind. Microbiol. Biotechnol 3:568-576; Svetina et al, 2000, J. Biotechnol 76: 245-251;Rasmussen-Wilson et al, 1997, Appl. Environ. Microbiol 63: 3488-3493;Ward et al, 1995, Biotechnology 13: 498-503; and Contreras et al., 1991,Biotechnology 9: 378-381; Eaton et al., 1986, Biochemistry 25: 505-512;Collins-Racie et al., 1995, Biotechnology 13: 982-987; Carter et al.,1989, Proteins: Structure, Function, and Genetics 6: 240-248; andStevens, 2003, Drug Discovery World 4: 35-48.

Sources of Polypeptides Having Catalase Activity

A polypeptide having catalase activity of the present invention may beobtained from microorganisms of any genus. For purposes of the presentinvention, the term “obtained from” as used herein in connection with agiven source shall mean that the polypeptide encoded by a polynucleotideis produced by the source or by a strain in which the polynucleotidefrom the source has been inserted. In one aspect, the polypeptideobtained from a given source is secreted extracellularly. Thepolypeptide may be a fungal polypeptide. For example, the polypeptidemay be a Malbranchea, Penicillium, or Rhizomucor, polypeptide.

In another aspect, the polypeptide is a Malbranchea cinnamomeapolypeptide. In another aspect, the polypeptide is a Rhizomucor pusilluspolypeptide. In another aspect, the polypeptide is a Penicilliumemersonii polypeptide. In another aspect, the polypeptide is aPenicillium funiculosum polypeptide. In another aspect, the polypeptideis a Penicillium purpurogenum polypeptide.

It will be understood that for the aforementioned species, the inventionencompasses both the perfect and imperfect states, and other taxonomicequivalents, e.g., anamorphs, regardless of the species name by whichthey are known. Those skilled in the art will readily recognize theidentity of appropriate equivalents.

Strains of these species are readily accessible to the public in anumber of culture collections, such as the American Type CultureCollection (ATCC), Deutsche Sammlung von Mikroorganismen andZellkulturen GmbH (DSMZ), Centraalbureau Voor Schimmelcultures (CBS),and Agricultural Research Service Patent Culture Collection, NorthernRegional Research Center (NRRL).

The polypeptide may be identified and obtained from other sourcesincluding microorganisms isolated from nature (e.g., soil, composts,water, etc.) or DNA samples obtained directly from natural materials(e.g., soil, composts, water, etc.) using the above-mentioned probes.Techniques for isolating microorganisms and DNA directly from naturalhabitats are well known in the art. A polynucleotide encoding thepolypeptide may then be obtained by similarly screening a genomic DNA orcDNA library of another microorganism or mixed DNA sample. Once apolynucleotide encoding a polypeptide has been detected with theprobe(s), the polynucleotide can be isolated or cloned by utilizingtechniques that are known to those of ordinary skill in the art (see,e.g., Sambrook et al., 1989, supra).

Catalytic Domains

In one embodiment, the present invention relates to catalytic domainshaving a sequence identity to amino acids 20 to 740 of SEQ ID NO: 8 ofat least 83%, e.g., at least 84%, at least 85%, at least 86%, at least87%, at least 88%, at least 89%, at least 90%, at least 91%, at least92%, at least 93%, at least 94%, at least 95%, at least 96%, at least97%, at least 98%, at least 99%, or 100%. In one embodiment, the presentinvention relates to catalytic domains having a sequence identity toamino acids 17 to 723 of SEQ ID NO: 2 of at least 76%, e.g., at least77%, at least 78%, at least 79%, at least 80%, at least 81%, at least82%, at least 83%, at least 84%, at least 85%, at least 86%, at least87%, at least 88%, at least 89%, at least 90%, at least 91%, at least92%, at least 93%, at least 94%, at least 95%, at least 96%, at least97%, at least 98%, at least 99%, or 100%. In one embodiment, the presentinvention relates to catalytic domains having a sequence identity toamino acids 38 to 723 of SEQ ID NO: 4 of at least 60%, e.g., at least65%, at least 70%, at least 75%, at least 78%, at least 80%, at least81%, at least 82%, at least 83%, at least 84%, at least 85%, at least86%, at least 87%, at least 88%, at least 89%, at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, at least 99%, or 100%. In oneembodiment, the present invention relates to catalytic domains having asequence identity to amino acids 38 to 711 of SEQ ID NO: 6 of at least60%, e.g., at least 65%, at least 70%, at least 75%, at least 78%, atleast 80%, at least 81%, at least 82%, at least 83%, at least 84%, atleast 85%, at least 86%, at least 87%, at least 88%, at least 89%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100%. In one aspect, the catalytic domains comprise amino acid sequencesthat differ by up to 10 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or10, from amino acids 20 to 740 of SEQ ID NO: 8, amino acids 17 to 723 ofSEQ ID NO: 2, amino acids 38 to 723 of SEQ ID NO: 4, or amino acids 38to 711 of SEQ ID NO: 6.

The catalytic domain preferably comprises or consists of amino acids 20to 740 of SEQ ID NO: 8 or an allelic variant thereof; or is a fragmentthereof having catalase activity. The catalytic domain preferablycomprises or consists of amino acids 17 to 723 of SEQ ID NO: 2 or anallelic variant thereof; or is a fragment thereof having catalaseactivity. The catalytic domain preferably comprises or consists of aminoacids 38 to 723 of SEQ ID NO: 4 or an allelic variant thereof; or is afragment thereof having catalase activity. The catalytic domainpreferably comprises or consists of amino acids 38 to 711 of SEQ ID NO:6 or an allelic variant thereof; or is a fragment thereof havingcatalase activity.

In another embodiment, the present invention relates to catalyticdomains encoded by polynucleotides that hybridize under very lowstringency conditions, low stringency conditions, medium stringencyconditions, medium-high stringency conditions, high stringencyconditions, and very high stringency conditions (as defined above) withnucleotides 58 to 2473 of SEQ ID NO: 7, nucleotides 49 to 2601 of SEQ IDNO: 1, nucleotides 112 to 2687 of SEQ ID NO: 3, or nucleotides 112 to2652 of SEQ ID NO: 5, or the full-length complement thereof (Sambrook etal., 1989, supra).

In another embodiment, the present invention relates to catalyticdomains encoded by polynucleotides having a sequence identity tonucleotides 58 to 2473 of SEQ ID NO: 7 of at least 83%, e.g., at least84%, at least 85%, at least 86%, at least 87%, at least 88%, at least89%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, at least99%, or 100%. In another embodiment, the present invention relates tocatalytic domains encoded by polynucleotides having a sequence identityto nucleotides 49 to 2601 of SEQ ID NO: 1 of at least 76%, e.g., atleast 77%, at least 78%, at least 79%, at least 80%, at least 81%, atleast 82%, at least 83%, at least 84%, at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100%. In another embodiment,the present invention relates to catalytic domains encoded bypolynucleotides having a sequence identity to nucleotides 112 to 2687 ofSEQ ID NO: 3 of at least 60%, e.g., at least 65%, at least 70%, at least75%, at least 78%, at least 80%, at least 81%, at least 82%, at least83%, at least 84%, at least 85%, at least 86%, at least 87%, at least88%, at least 89%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, at least 99%, or 100%. In another embodiment, the present inventionrelates to catalytic domains encoded by polynucleotides having asequence identity to nucleotides 112 to 2652 of SEQ ID NO: 5 of at least60%, e.g., at least 65%, at least 70%, at least 75%, at least 78%, atleast 80%, at least 81%, at least 82%, at least 83%, at least 84%, atleast 85%, at least 86%, at least 87%, at least 88%, at least 89%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100%.

The polynucleotide encoding the catalytic domain preferably comprises orconsists of nucleotides 58 to 2473 of SEQ ID NO: 7. The polynucleotideencoding the catalytic domain preferably comprises or consists ofnucleotides 49 to 2601 of SEQ ID NO: 1. The polynucleotide encoding thecatalytic domain preferably comprises or consists of nucleotides 112 to2687 of SEQ ID NO: 3. The polynucleotide encoding the catalytic domainpreferably comprises or consists of nucleotides 112 to 2652 of SEQ IDNO: 5.

In another embodiment, the present invention relates to catalytic domainvariants of amino acids 20 to 740 of SEQ ID NO: 8, catalytic domainvariants of amino acids 17 to 723 of SEQ ID NO: 2, catalytic domainvariants of amino acids 38 to 723 of SEQ ID NO: 4 or catalytic domainvariants of amino acids 38 to 711 of SEQ ID NO: 6, comprising asubstitution, deletion, and/or insertion at one or more (e.g., several)positions. In one aspect, the number of amino acid substitutions,deletions and/or insertions introduced into the sequence of amino acids20 to 740 of SEQ ID NO: 8, amino acids 17 to 723 of SEQ ID NO: 2, aminoacids 38 to 723 of SEQ ID NO: 4, or amino acids 38 to 711 of SEQ ID NO:6, is up to 10, e.g., 1, 2, 3, 4, 5, 6, 8, 9, or 10.

Polynucleotides

The present invention also relates to isolated polynucleotides encodinga polypeptide, a catalytic domain of the present invention, as describedherein.

The techniques used to isolate or clone a polynucleotide are known inthe art and include isolation from genomic DNA or cDNA, or a combinationthereof. The cloning of the polynucleotides from genomic DNA can beeffected, e.g., by using the well known polymerase chain reaction (PCR)or antibody screening of expression libraries to detect cloned DNAfragments with shared structural features. See, e.g., Innis et al, 1990,PCR: A Guide to Methods and Application, Academic Press, New York. Othernucleic acid amplification procedures such as ligase chain reaction(LCR), ligation activated transcription (LAT) and polynucleotide-basedamplification (NASBA) may be used. The polynucleotides may be clonedfrom a strain of Malbranchea, Rhizomucor or Penicillium, or a relatedorganism and thus, for example, may be an allelic or species variant ofthe polypeptide encoding region of the polynucleotide.

Modification of a polynucleotide encoding a polypeptide of the presentinvention may be necessary for synthesizing polypeptides substantiallysimilar to the polypeptide. The term “substantially similar” to thepolypeptide refers to non-naturally occurring forms of the polypeptide.These polypeptides may differ in some engineered way from thepolypeptide isolated from its native source, e.g., variants that differin specific activity, thermostability, pH optimum, or the like. Thevariants may be constructed on the basis of the polynucleotide presentedas the mature polypeptide coding sequence of SEQ ID NO: 7, the maturepolypeptide coding sequence of SEQ ID NO: 1, the mature polypeptidecoding sequence of SEQ ID NO: 3, or the mature polypeptide codingsequence of SEQ ID NO: 5, or the cDNA sequence thereof, or a subsequencethereof, by introduction of nucleotide substitutions that do not resultin a change in the amino acid sequence of the polypeptide, but whichcorrespond to the codon usage of the host organism intended forproduction of the enzyme, or by introduction of nucleotide substitutionsthat may give rise to a different amino acid sequence. For a generaldescription of nucleotide substitution, see, e.g., Ford et al, 1991,Protein Expression and Purification 2: 95-107.

Nucleic Acid Constructs

The present invention also relates to nucleic acid constructs comprisinga polynucleotide of the present invention operably linked to one or morecontrol (e.g., several) sequences that direct the expression of thecoding sequence in a suitable host cell under conditions compatible withthe control sequences.

The polynucleotide may be manipulated in a variety of ways to providefor expression of the polypeptide. Manipulation of the polynucleotideprior to its insertion into a vector may be desirable or necessarydepending on the expression vector. The techniques for modifyingpolynucleotides utilizing recombinant DNA methods are well known in theart.

The control sequence may be a promoter, a polynucleotide that isrecognized by a host cell for expression of a polynucleotide encoding apolypeptide of the present invention. The promoter containstranscriptional control sequences that mediate the expression of thepolypeptide. The promoter may be any polynucleotide that showstranscriptional activity in the host cell including mutant, truncated,and hybrid promoters, and may be obtained from genes encodingextracellular or intracellular polypeptides either homologous orheterologous to the host cell.

Examples of suitable promoters for directing transcription of thenucleic acid constructs of the present invention in a bacterial hostcell are the promoters obtained from the Bacillus amyloliquefaciensalpha-amylase gene (amyQ), Bacillus licheniformis alpha-amylase gene(amyL), Bacillus licheniformis penicillinase gene (penP), Bacillusstearothermophilus maltogenic amylase gene (amyM), Bacillus subtilislevansucrase gene (sacB), Bacillus subtilis xylA and xylB genes,Bacillus thuringiensis crylllA gene (Agaisse and Lereclus, 1994,Molecular Microbiology 13: 97-107), E. coli lac operon, E. coli trcpromoter (Egon et al., 1988, Gene 69: 301-315), Streptomyces coelicoloragarase gene (dagA), and prokaryotic beta-lactamase gene (Villa-Kamaroffet al., 1978, Proc. Natl. Acad. Sci. USA 75: 3727-3731), as well as thetac promoter (DeBoer et al., 1983, Proc. Natl. Acad. Sci. USA 80:21-25). Further promoters are described in “Useful proteins fromrecombinant bacteria” in Gilbert et al., 1980, Scientific American 242:74-94; and in Sambrook et al., 1989, supra. Examples of tandem promotersare disclosed in WO 99/43835.

Examples of suitable promoters for directing transcription of thenucleic acid constructs of the present invention in a filamentous fungalhost cell are promoters obtained from the genes for Aspergillus nidulansacetamidase, Aspergillus niger neutral alpha-amylase, Aspergillus nigeracid stable alpha-amylase, Aspergillus niger or Aspergillus awamoriglucoamylase (glaA), Aspergillus oryzae TAKA amylase, Aspergillus oryzaealkaline protease, Aspergillus oryzae triose phosphate isomerase,Fusarium oxysporum trypsin-like protease (WO 96/00787), Fusariumvenenatum amyloglucosidase (WO 00/56900), Fusarium venenatum Dania (WO00/56900), Fusarium venenatum Quinn (WO 00/56900), Rhizomucor mieheilipase, Rhizomucor miehei aspartic proteinase, Trichoderma reeseibeta-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichodermareesei cellobiohydrolase II, Trichoderma reesei endoglucanase I,Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanaseIII, Trichoderma reesei endoglucanase V, Trichoderma reesei xylanase I,Trichoderma reesei xylanase II, Trichoderma reesei xylanase III,Trichoderma reesei beta-xylosidase, and Trichoderma reesei translationelongation factor, as well as the NA2-tpi promoter (a modified promoterfrom an Aspergillus neutral alpha-amylase gene in which the untranslatedleader has been replaced by an untranslated leader from an Aspergillustriose phosphate isomerase gene; non-limiting examples include modifiedpromoters from an Aspergillus niger neutral alpha-amylase gene in whichthe untranslated leader has been replaced by an untranslated leader froman Aspergillus nidulans or Aspergillus oryzae triose phosphate isomerasegene); and mutant, truncated, and hybrid promoters thereof. Otherpromoters are described in U.S. Pat. No. 6,011,147.

In a yeast host, useful promoters are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiaegalactokinase (GAL1), Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH1, ADH2/GAP),Saccharomyces cerevisiae triose phosphate isomerase (TPI), Saccharomycescerevisiae metallothionein (CUP1), and Saccharomyces cerevisiae3-phosphoglycerate kinase. Other useful promoters for yeast host cellsare described by Romanos et al, 1992, Yeast 8: 423-488.

The control sequence may also be a transcription terminator, which isrecognized by a host cell to terminate transcription. The terminator isoperably linked to the 3′-terminus of the polynucleotide encoding thepolypeptide. Any terminator that is functional in the host cell may beused in the present invention.

Preferred terminators for bacterial host cells are obtained from thegenes for Bacillus clausii alkaline protease (aprH), Bacilluslicheniformis alpha-amylase (amyL), and Escherichia coli ribosomal RNA(rmB).

Preferred terminators for filamentous fungal host cells are obtainedfrom the genes for Aspergillus nidulans acetamidase, Aspergillusnidulans anthranilate synthase, Aspergillus niger glucoamylase,Aspergillus niger alpha-glucosidase, Aspergillus oryzae TAKA amylase,Fusarium oxysporum trypsin-like protease, Trichoderma reeseibeta-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichodermareesei cellobiohydrolase II, Trichoderma reesei endoglucanase I,Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanaseIII, Trichoderma reesei endoglucanase V, Trichoderma reesei xylanase I,Trichoderma reesei xylanase II, Trichoderma reesei xylanase III,Trichoderma reesei beta-xylosidase, and Trichoderma reesei translationelongation factor.

Preferred terminators for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae enolase, Saccharomyces cerevisiaecytochrome C (CYC1), and Saccharomyces cerevisiaeglyceraldehyde-3-phosphate dehydrogenase. Other useful terminators foryeast host cells are described by Romanos et al., 1992, supra.

The control sequence may also be an mRNA stabilizer region downstream ofa promoter and upstream of the coding sequence of a gene which increasesexpression of the gene.

Examples of suitable mRNA stabilizer regions are obtained from aBacillus thuringiensis cryIIIA gene (WO 94/25612) and a Bacillussubtilis SP82 gene (Hue et al, 1995, Journal of Bacteriology 177:3465-3471).

The control sequence may also be a leader, a nontranslated region of anmRNA that is important for translation by the host cell. The leader isoperably linked to the 5′-terminus of the polynucleotide encoding thepolypeptide. Any leader that is functional in the host cell may be used.

Preferred leaders for filamentous fungal host cells are obtained fromthe genes for Aspergillus otyzae TAKA amylase and Aspergillus nidulanstriose phosphate isomerase.

Suitable leaders for yeast host cells are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, andSaccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).

The control sequence may also be a polyadenylation sequence, a sequenceoperably linked to the 3′-terminus of the polynucleotide and, whentranscribed, is recognized by the host cell as a signal to addpolyadenosine residues to transcribed mRNA. Any polyadenylation sequencethat is functional in the host cell may be used.

Preferred polyadenylation sequences for filamentous fungal host cellsare obtained from the genes for Aspergillus nidulans anthranilatesynthase, Aspergillus niger glucoamylase, Aspergillus nigeralpha-glucosidase Aspergillus oryzae TAKA amylase, and Fusariumoxysporum trypsin-like protease.

Useful polyadenylation sequences for yeast host cells are described byGuo and Sherman, 1995, Mol. Cellular Biol. 15: 5983-5990.

The control sequence may also be a signal peptide coding region thatencodes a signal peptide linked to the N-terminus of a polypeptide anddirects the polypeptide into the cell's secretory pathway. The 5′-end ofthe coding sequence of the polynucleotide may inherently contain asignal peptide coding sequence naturally linked in translation readingframe with the segment of the coding sequence that encodes thepolypeptide. Alternatively, the 5′-end of the coding sequence maycontain a signal peptide coding sequence that is foreign to the codingsequence. A foreign signal peptide coding sequence may be required wherethe coding sequence does not naturally contain a signal peptide codingsequence. Alternatively, a foreign signal peptide coding sequence maysimply replace the natural signal peptide coding sequence in order toenhance secretion of the polypeptide. However, any signal peptide codingsequence that directs the expressed polypeptide into the secretorypathway of a host cell may be used.

Effective signal peptide coding sequences for bacterial host cells arethe signal peptide coding sequences obtained from the genes for BacillusNCIB 11837 maltogenic amylase, Bacillus licheniformis subtilisin,Bacillus licheniformis beta-lactamase, Bacillus stearothermophilusalpha-amylase, Bacillus stearothermophilus neutral proteases (nprT,nprS, nprM), and Bacillus subtilis prsA. Further signal peptides aredescribed by Simonen and Palva, 1993, Microbiological Reviews 57:109-137.

Effective signal peptide coding sequences for filamentous fungal hostcells are the signal peptide coding sequences obtained from the genesfor Aspergillus niger neutral amylase, Aspergillus niger glucoamylase,Aspergillus oryzae TAKA amylase, Humicola insolens cellulase,

Humicola insolens endoglucanase V, Humicola lanuginosa lipase, andRhizomucor miehei aspartic proteinase.

Useful signal peptides for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiaeinvertase. Other useful signal peptide coding sequences are described byRomanos et al., 1992, supra.

The control sequence may also be a propeptide coding sequence thatencodes a propeptide positioned at the N-terminus of a polypeptide. Theresultant polypeptide is known as a proenzyme or propolypeptide (or azymogen in some cases). A propolypeptide is generally inactive and canbe converted to an active polypeptide by catalytic or autocatalyticcleavage of the propeptide from the propolypeptide. The propeptidecoding sequence may be obtained from the genes for Bacillus subtilisalkaline protease (aprE), Bacillus subtilis neutral protease (nprT),Myceliophthora thermophila laccase (WO 95/33836), Rhizomucor mieheiaspartic proteinase, and Saccharomyces cerevisiae alpha-factor.

Where both signal peptide and propeptide sequences are present, thepropeptide sequence is positioned next to the N-terminus of apolypeptide and the signal peptide sequence is positioned next to theN-terminus of the propeptide sequence.

It may also be desirable to add regulatory sequences that regulateexpression of the polypeptide relative to the growth of the host cell.Examples of regulatory sequences are those that cause expression of thegene to be turned on or off in response to a chemical or physicalstimulus, including the presence of a regulatory compound. Regulatorysequences in prokaryotic systems include the lac, tac, and trp operatorsystems. In yeast, the ADH2 system or GAL1 system may be used. Infilamentous fungi, the Aspergillus niger glucoamylase promoter,Aspergillus oryzae TAKA alpha-amylase promoter, and Aspergillus oryzaeglucoamylase promoter, Trichoderma reesei cellobiohydrolase I promoter,and Trichoderma reesei cellobiohydrolase II promoter may be used. Otherexamples of regulatory sequences are those that allow for geneamplification. In eukaryotic systems, these regulatory sequences includethe dihydrofolate reductase gene that is amplified in the presence ofmethotrexate, and the metallothionein genes that are amplified withheavy metals. In these cases, the polynucleotide encoding thepolypeptide would be operably linked with the regulatory sequence.

Expression Vectors

The present invention also relates to recombinant expression vectorscomprising a polynucleotide of the present invention, a promoter, andtranscriptional and translational stop signals. The various nucleotideand control sequences may be joined together to produce a recombinantexpression vector that may include one or more (e.g., several)convenient restriction sites to allow for insertion or substitution ofthe polynucleotide encoding the polypeptide at such sites.Alternatively, the polynucleotide may be expressed by inserting thepolynucleotide or a nucleic acid construct comprising the polynucleotideinto an appropriate vector for expression. In creating the expressionvector, the coding sequence is located in the vector so that the codingsequence is operably linked with the appropriate control sequences forexpression.

The recombinant expression vector may be any vector (e.g., a plasmid orvirus) that can be conveniently subjected to recombinant DNA proceduresand can bring about expression of the polynucleotide. The choice of thevector will typically depend on the compatibility of the vector with thehost cell into which the vector is to be introduced. The vector may be alinear or closed circular plasmid.

The vector may be an autonomously replicating vector, i.e., a vectorthat exists as an extrachromosomal entity, the replication of which isindependent of chromosomal replication, e.g., a plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.The vector may contain any means for assuring self-replication.Alternatively, the vector may be one that, when introduced into the hostcell, is integrated into the genome and replicated together with thechromosome(s) into which it has been integrated. Furthermore, a singlevector or plasmid or two or more vectors or plasmids that togethercontain the total DNA to be introduced into the genome of the host cell,or a transposon, may be used.

The vector preferably contains one or more (e.g., several) selectablemarkers that permit easy selection of transformed, transfected,transduced, or the like cells. A selectable marker is a gene the productof which provides for biocide or viral resistance, resistance to heavymetals, prototrophy to auxotrophs, and the like.

Examples of bacterial selectable markers are Bacillus licheniformis orBacillus subtilis dal genes, or markers that confer antibioticresistance such as ampicillin, chloramphenicol, kanamycin, neomycin,spectinomycin, or tetracycline resistance. Suitable markers for yeasthost cells include, but are not limited to, ADE2, HIS3, LEU2, LYS2,MET3, TRP1, and URA3. Selectable markers for use in a filamentous fungalhost cell include, but are not limited to, adeA(phosphoribosylaminoimidazole-succinocarboxamide synthase), adeB(phosphoribosyl-aminoimidazole synthase), amdS (acetamidase), argB(ornithine carbamoyltransferase), bar (phosphinothricinacetyltransferase), hph (hygromycin phosphotransferase), niaD (nitratereductase), pyrG (orotidine-5′-phosphate decarboxylase), sC (sulfateadenyltransferase), and trpC (anthranilate synthase), as well asequivalents thereof. Preferred for use in an Aspergillus cell areAspergillus nidulans or Aspergillus oryzae amdS and pyrG genes and aStreptomyces hygroscopicus bar gene. Preferred for use in a Trichodermacell are adeA, adeB, amdS, hph, and pyrG genes.

The selectable marker may be a dual selectable marker system asdescribed in WO 2010/039889. In one aspect, the dual selectable markeris a hph-tk dual selectable marker system.

The vector preferably contains an element(s) that permits integration ofthe vector into the host cell's genome or autonomous replication of thevector in the cell independent of the genome.

For integration into the host cell genome, the vector may rely on thepolynucleotide's sequence encoding the polypeptide or any other elementof the vector for integration into the genome by homologous ornon-homologous recombination. Alternatively, the vector may containadditional polynucleotides for directing integration by homologousrecombination into the genome of the host cell at a precise location(s)in the chromosome(s). To increase the likelihood of integration at aprecise location, the integrational elements should contain a sufficientnumber of nucleic acids, such as 100 to 10,000 base pairs, 400 to 10,000base pairs, and 800 to 10,000 base pairs, which have a high degree ofsequence identity to the corresponding target sequence to enhance theprobability of homologous recombination. The integrational elements maybe any sequence that is homologous with the target sequence in thegenome of the host cell. Furthermore, the integrational elements may benon-encoding or encoding polynucleotides. On the other hand, the vectormay be integrated into the genome of the host cell by non-homologousrecombination.

For autonomous replication, the vector may further comprise an origin ofreplication enabling the vector to replicate autonomously in the hostcell in question. The origin of replication may be any plasmidreplicator mediating autonomous replication that functions in a cell.The term “origin of replication” or “plasmid replicator” means apolynucleotide that enables a plasmid or vector to replicate in vivo.

Examples of bacterial origins of replication are the origins ofreplication of plasmids pBR322, pUC19, pACYC177, and pACYC184 permittingreplication in E. coli, and pUB110, pE194, pTA1060, and pAMβ111permitting replication in Bacillus.

Examples of origins of replication for use in a yeast host cell are the2 micron origin of replication, ARS1, ARS4, the combination of ARS1 andCEN3, and the combination of ARS4 and CEN6.

Examples of origins of replication useful in a filamentous fungal cellare AMA1 and ANSI (Gems et al, 1991, Gene 98: 61-67; Cullen et al, 1987,Nucleic Acids Res. 15: 9163-9175; WO 00/24883). Isolation of the AMA1gene and construction of plasmids or vectors comprising the gene can beaccomplished according to the methods disclosed in WO 00/24883.

More than one copy of a polynucleotide of the present invention may beinserted into a host cell to increase production of a polypeptide. Anincrease in the copy number of the polynucleotide can be obtained byintegrating at least one additional copy of the sequence into the hostcell genome or by including an amplifiable selectable marker gene withthe polynucleotide where cells containing amplified copies of theselectable marker gene, and thereby additional copies of thepolynucleotide, can be selected for by cultivating the cells in thepresence of the appropriate selectable agent.

The procedures used to ligate the elements described above to constructthe recombinant expression vectors of the present invention are wellknown to one skilled in the art (see, e.g., Sambrook et al, 1989,supra).

Host Cells

The present invention also relates to recombinant host cells, comprisinga polynucleotide of the present invention operably linked to one or more(e.g., several) control sequences that direct the production of apolypeptide of the present invention. A construct or vector comprising apolynucleotide is introduced into a host cell so that the construct orvector is maintained as a chromosomal integrant or as a self-replicatingextra-chromosomal vector as described earlier. The term “host cell”encompasses any progeny of a parent cell that is not identical to theparent cell due to mutations that occur during replication. The choiceof a host cell will to a large extent depend upon the gene encoding thepolypeptide and its source.

The host cell may be any cell useful in the recombinant production of apolypeptide of the present invention, e.g., a prokaryote or a eukaryote.

The prokaryotic host cell may be any Gram-positive or Gram-negativebacterium. Gram-positive bacteria include, but are not limited to,Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus,Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, andStreptomyces. Gram-negative bacteria include, but are not limited to,Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter,Ilyobacter, Neisseria, Pseudomonas, Salmonella, and Ureaplasma.

The bacterial host cell may be any Bacillus cell including, but notlimited to, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillusbrevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans,Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacilluslicheniformis, Bacillus megaterium, Bacillus pumilus, Bacillusstearothermophilus, Bacillus subtilis, and Bacillus thuringiensis cells.

The bacterial host cell may also be any Streptococcus cell including,but not limited to, Streptococcus equisimilis, Streptococcus pyogenes,Streptococcus uberis, and Streptococcus equi subsp. Zooepidemicus cells.

The bacterial host cell may also be any Streptomyces cell including, butnot limited to, Streptomyces achromogenes, Streptomyces avermitilis,Streptomyces coelicolor, Streptomyces griseus, and Streptomyces lividanscells.

The introduction of DNA into a Bacillus cell may be effected byprotoplast transformation (see, e.g., Chang and Cohen, 1979, Mol. Gen.Genet. 168: 111-115), competent cell transformation (see, e.g., Youngand Spizizen, 1961, J. Bacteriol. 81: 823-829, or Dubnau andDavidoff-Abelson, 1971, J. Mol. Biol. 56: 209-221), electroporation(see, e.g., Shigekawa and Dower, 1988, Biotechniques 6: 742-751), orconjugation (see, e.g., Koehler and Thorne, 1987, J. Bacteriol. 169:5271-5278). The introduction of DNA into an E. coli cell may be effectedby protoplast transformation (see, e.g., Hanahan, 1983, J. Mol. Biol.166: 557-580) or electroporation (see, e.g., Dower et al, 1988, NucleicAcids Res. 16: 6127-6145). The introduction of DNA into a Streptomycescell may be effected by protoplast transformation, electroporation (see,e.g., Gong et al., 2004, Folia Microbiol (Praha) 49: 399-405),conjugation (see, e.g., Mazodier et al, 1989, J. Bacteriol. 171:3583-3585), or transduction (see, e.g., Burke et al, 2001, Proc. Natl.Acad. Sci. USA 98: 6289-6294). The introduction of DNA into aPseudomonas cell may be effected by electroporation (see, e.g., Choi etal, 2006, J. Microbiol. Methods 64: 391-397) or conjugation (see, e.g.,Pinedo and Smets, 2005, Appl. Environ. Microbiol. 71: 51-57). Theintroduction of DNA into a Streptococcus cell may be effected by naturalcompetence (see, e.g., Perry and Kuramitsu, 1981, Infect. Immun. 32:1295-1297), protoplast transformation (see, e.g., Catt and Jollick,1991, Microbios 68: 189-207), electroporation (see, e.g., Buckley etal., 1999, Appl. Environ. Microbiol 65: 3800-3804), or conjugation (see,e.g., Clewell, 1981, Microbiol. Rev. 45: 409-436). However, any methodknown in the art for introducing DNA into a host cell can be used.

The host cell may also be a eukaryote, such as a mammalian, insect,plant, or fungal cell.

The host cell may be a fungal cell. “Fungi” as used herein includes thephyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota as wellas the Oomycota and all mitosporic fungi (as defined by Hawksworth etal., In, Ainsworth and Bisby's Dictionary of The Fungi, 8th edition,1995, CAB International, University Press, Cambridge, UK).

The fungal host cell may be a yeast cell. “Yeast” as used hereinincludes ascosporogenous yeast (Endomycetales), basidiosporogenousyeast, and yeast belonging to the Fungi Imperfecti (Blastomycetes).Since the classification of yeast may change in the future, for thepurposes of this invention, yeast shall be defined as described inBiology and Activities of Yeast (Skinner, Passmore, and Davenport,editors, Soc. App. Bacteriol. Symposium Series No. 9, 1980).

The yeast host cell may be a Candida, Hansenula, Kluyveromyces, Pichia,Saccharomyces, Schizosaccharomyces, or Yarrowia cell, such as aKluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomycescerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii,Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomycesoviformis, or Yarrowia lipolytica cell.

The fungal host cell may be a filamentous fungal cell. “Filamentousfungi” include all filamentous forms of the subdivision Eumycota andOomycota (as defined by Hawksworth et al., 1995, supra). The filamentousfungi are generally characterized by a mycelial wall composed of chitin,cellulose, glucan, chitosan, mannan, and other complex polysaccharides.Vegetative growth is by hyphal elongation and carbon catabolism isobligately aerobic. In contrast, vegetative growth by yeasts such asSaccharomyces cerevisiae is by budding of a unicellular thallus andcarbon catabolism may be fermentative.

The filamentous fungal host cell may be an Acremonium, Aspergillus,Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus,Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe,Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces,Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus,Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium,Trametes, or Trichoderma cell.

For example, the filamentous fungal host cell may be an Aspergillusawamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillusjaponicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae,Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis caregiea,Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsisrivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora,Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporiumlucknowense, Chrysosporium merdarium, Chrysosporium pannicola,Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporiumzonatum, Coprinus cinereus, Coriolus hirsutus, Fusarium bactridioides,Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusariumgraminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi,Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusariumsambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusariumsulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusariumvenenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei,Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum,Phanerochaete chrysosporium, Phlebia radiata, Pleurotus etyngii,Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichodermahatzianum, Trichoderma koningii, Trichoderma longibrachiatum,Trichoderma reesei, or Trichoderma viride cell.

Fungal cells may be transformed by a process involving protoplastformation, transformation of the protoplasts, and regeneration of thecell wall in a manner known per se. Suitable procedures fortransformation of Aspergillus and Trichoderma host cells are describedin EP 238023, Yelton et al., 1984, Proc. Natl. Acad. Sci. USA 81:1470-1474, and Christensen et al., 1988, Bio/Technology 6: 1419-1422.Suitable methods for transforming Fusarium species are described byMalardier et al., 1989, Gene 78: 147-156, and WO 96/00787. Yeast may betransformed using the procedures described by Becker and Guarente, InAbelson, J. N. and Simon, M. I., editors, Guide to Yeast Genetics andMolecular Biology, Methods in Enzymology, Volume 194, pp 182-187,Academic Press, Inc., New York; Ito et al., 1983, J. Bacteriol 153: 163;and Hinnen et al., 1978, Proc. Natl. Acad. Sci. USA 75: 1920.

Methods of Production

The present invention also relates to methods of producing a polypeptideof the present invention, comprising (a) cultivating a cell, which inits wild-type form produces the polypeptide, under conditions conducivefor production of the polypeptide; and optionally (b) recovering thepolypeptide. In a preferred aspect, the cell is a Malbranchea cell. In amore preferred aspect, the cell is a Malbranchea cinnamomea cell. In amost preferred aspect, the cell is Malbranchea cinnamomea NN044758. In apreferred aspect, the cell is a Rhizomucor cell. In a more preferredaspect, the cell is a Rhizomucor pusillus cell. In a most preferredaspect, the cell is Rhizomucor pusillus NN046782. In another aspect, thecell is a Penicillium cell. In another aspect, the cell is a Penicilliumemersonii cell. In another aspect, the cell is a Penicillium emersoniiNNO51602.

The present invention also relates to methods of producing a polypeptideof the present invention, comprising (a) cultivating a recombinant hostcell of the present invention under conditions conducive for productionof the polypeptide; and optionally (b) recovering the polypeptide.

The host cells are cultivated in a nutrient medium suitable forproduction of the polypeptide using methods known in the art. Forexample, the cells may be cultivated by shake flask cultivation, orsmall-scale or large-scale fermentation (including continuous, batch,fed-batch, or solid state fermentations) in laboratory or industrialfermentors in a suitable medium and under conditions allowing thepolypeptide to be expressed and/or isolated. The cultivation takes placein a suitable nutrient medium comprising carbon and nitrogen sources andinorganic salts, using procedures known in the art. Suitable media areavailable from commercial suppliers or may be prepared according topublished compositions (e.g., in catalogues of the American Type CultureCollection). If the polypeptide is secreted into the nutrient medium,the polypeptide can be recovered directly from the medium. If thepolypeptide is not secreted, it can be recovered from cell lysates.

The polypeptide may be detected using methods known in the art that arespecific for the polypeptides. These detection methods include, but arenot limited to, use of specific antibodies, formation of an enzymeproduct, or disappearance of an enzyme substrate. For example, an enzymeassay may be used to determine the activity of the polypeptide.

The polypeptide may be recovered using methods known in the art. Forexample, the polypeptide may be recovered from the nutrient medium byconventional procedures including, but not limited to, collection,centrifugation, filtration, extraction, spray-drying, evaporation, orprecipitation. In one aspect, a whole fermentation broth comprising thepolypeptide is recovered.

The polypeptide may be purified by a variety of procedures known in theart including, but not limited to, chromatography (e.g., ion exchange,affinity, hydrophobic, chromatofocusing, and size exclusion),electrophoretic procedures (e.g., preparative isoelectric focusing),differential solubility (e.g., ammonium sulfate precipitation),SDS-PAGE, or extraction (see, e.g., Protein Purification, Janson andRyden, editors, VCH Publishers, New York, 1989) to obtain substantiallypure polypeptides.

In an alternative aspect, the polypeptide is not recovered, but rather ahost cell of the present invention expressing the polypeptide is used asa source of the polypeptide.

Plants

The present invention also relates to isolated plants, e.g., atransgenic plant, plant part, or plant cell, comprising a polynucleotideof the present invention so as to express and produce a polypeptide ordomain in recoverable quantities. The polypeptide or domain may berecovered from the plant or plant part. Alternatively, the plant orplant part containing the polypeptide or domain may be used as such forimproving the quality of a food or feed, e.g., improving nutritionalvalue, palatability, and rheological properties, or to destroy anantinutritive factor.

The transgenic plant can be dicotyledonous (a dicot) or monocotyledonous(a monocot). Examples of monocot plants are grasses, such as meadowgrass (blue grass, Poa), forage grass such as Festuca, Lolium, temperategrass, such as Agrostis, and cereals, e.g., wheat, oats, rye, barley,rice, sorghum, and maize (corn).

Examples of dicot plants are tobacco, legumes, such as lupins, potato,sugar beet, pea, bean and soybean, and cruciferous plants (familyBrassicaceae), such as cauliflower, rape seed, and the closely relatedmodel organism Arabidopsis thaliana.

Examples of plant parts are stem, callus, leaves, root, fruits, seeds,and tubers as well as the individual tissues comprising these parts,e.g., epidermis, mesophyll, parenchyme, vascular tissues, meristems.Specific plant cell compartments, such as chloroplasts, apoplasts,mitochondria, vacuoles, peroxisomes and cytoplasm are also considered tobe a plant part. Furthermore, any plant cell, whatever the tissueorigin, is considered to be a plant part. Likewise, plant parts such asspecific tissues and cells isolated to facilitate the utilization of theinvention are also considered plant parts, e.g., embryos, endosperms,aleurone and seed coats.

Also included within the scope of the present invention are the progenyof such plants, plant parts, and plant cells.

The transgenic plant or plant cell expressing the polypeptide or domainmay be constructed in accordance with methods known in the art. Inshort, the plant or plant cell is constructed by incorporating one ormore expression constructs encoding the polypeptide or domain into theplant host genome or chloroplast genome and propagating the resultingmodified plant or plant cell into a transgenic plant or plant cell.

The expression construct is conveniently a nucleic acid construct thatcomprises a polynucleotide encoding a polypeptide or domain operablylinked with appropriate regulatory sequences required for expression ofthe polynucleotide in the plant or plant part of choice. Furthermore,the expression construct may comprise a selectable marker useful foridentifying plant cells into which the expression construct has beenintegrated and DNA sequences necessary for introduction of the constructinto the plant in question (the latter depends on the DNA introductionmethod to be used).

The choice of regulatory sequences, such as promoter and terminatorsequences and optionally signal or transit sequences, is determined, forexample, on the basis of when, where, and how the polypeptide or domainis desired to be expressed. For instance, the expression of the geneencoding a polypeptide or domain may be constitutive or inducible, ormay be developmental, stage or tissue specific, and the gene product maybe targeted to a specific tissue or plant part such as seeds or leaves.Regulatory sequences are, for example, described by Tague et al., 1988,Plant Physiology 86: 506.

For constitutive expression, the 35S-CaMV, the maize ubiquitin 1, or therice actin 1 promoter may be used (Franck et al., 1980, Cell 21:285-294; Christensen et al., 1992, Plant Mol. Biol. 18: 675-689; Zhanget al., 1991, Plant Cell 3: 1155-1165). Organ-specific promoters may be,for example, a promoter from storage sink tissues such as seeds, potatotubers, and fruits (Edwards and Coruzzi, 1990, Ann. Rev. Genet. 24:275-303), or from metabolic sink tissues such as meristems (Ito et al.,1994, Plant Mol. Biol. 24: 863-878), a seed specific promoter such asthe glutelin, prolamin, globulin, or albumin promoter from rice (Wu etal., 1998, Plant Cell PhysioL 39: 885-889), a Vicia faba promoter fromthe legumin B4 and the unknown seed protein gene from Vicia faba (Conradet al, 1998, J. Plant Physiol 152: 708-711), a promoter from a seed oilbody protein (Chen et al., 1998, Plant Cell Physiol 39: 935-941), thestorage protein napA promoter from Brassica napus, or any other seedspecific promoter known in the art, e.g., as described in WO 91/14772.Furthermore, the promoter may be a leaf specific promoter such as therbcs promoter from rice or tomato (Kyozuka et al, 1993, Plant Physiol102: 991-1000), the chlorella virus adenine methyltransferase genepromoter (Mitra and Higgins, 1994, Plant Mol. Biol. 26: 85-93), the aldPgene promoter from rice (Kagaya et al., 1995, Mol. Gen. Genet. 248:668-674), or a wound inducible promoter such as the potato pin2 promoter(Xu et al., 1993, Plant Mol. Biol. 22: 573-588). Likewise, the promotermay be induced by abiotic treatments such as temperature, drought, oralterations in salinity or induced by exogenously applied substancesthat activate the promoter, e.g., ethanol, oestrogens, plant hormonessuch as ethylene, abscisic acid, and gibberellic acid, and heavy metals.

A promoter enhancer element may also be used to achieve higherexpression of a polypeptide or domain in the plant. For instance, thepromoter enhancer element may be an intron that is placed between thepromoter and the polynucleotide encoding a polypeptide or domain. Forinstance, Xu et al, 1993, supra, disclose the use of the first intron ofthe rice actin 1 gene to enhance expression.

The selectable marker gene and any other parts of the expressionconstruct may be chosen from those available in the art.

The nucleic acid construct is incorporated into the plant genomeaccording to conventional techniques known in the art, includingAgrobacterium-mediated transformation, virus-mediated transformation,microinjection, particle bombardment, biolistic transformation, andelectroporation (Gasser et al., 1990, Science 244: 1293; Potrykus, 1990,Bio/Technology 8: 535; Shimamoto et al, 1989, Nature 338: 274).

Agrobacterium tumefaciens-mediated gene transfer is a method forgenerating transgenic dicots (for a review, see Hooykas andSchilperoort, 1992, Plant Mol. Biol. 19: 15-38) and for transformingmonocots, although other transformation methods may be used for theseplants. A method for generating transgenic monocots is particlebombardment (microscopic gold or tungsten particles coated with thetransforming DNA) of embryonic calli or developing embryos (Christou,1992, Plant J. 2: 275-281; Shimamoto, 1994, Curr. Opin. Biotechnol. 5:158-162; Vasil et al., 1992, Bio/Technology 10: 667-674). An alternativemethod for transformation of monocots is based on protoplasttransformation as described by Omirulleh et al, 1993, Plant Mol. Biol.21: 415-428. Additional transformation methods include those describedin U.S. Pat. Nos. 6,395,966 and 7,151,204 (both of which are hereinincorporated by reference in their entirety).

Following transformation, the transformants having incorporated theexpression construct are selected and regenerated into whole plantsaccording to methods well known in the art. Often the transformationprocedure is designed for the selective elimination of selection geneseither during regeneration or in the following generations by using, forexample, co-transformation with two separate T-DNA constructs or sitespecific excision of the selection gene by a specific recombinase.

In addition to direct transformation of a particular plant genotype witha construct of the present invention, transgenic plants may be made bycrossing a plant having the construct to a second plant lacking theconstruct. For example, a construct encoding a polypeptide or domain canbe introduced into a particular plant variety by crossing, without theneed for ever directly transforming a plant of that given variety.Therefore, the present invention encompasses not only a plant directlyregenerated from cells which have been transformed in accordance withthe present invention, but also the progeny of such plants. As usedherein, progeny may refer to the offspring of any generation of a parentplant prepared in accordance with the present invention. Such progenymay include a DNA construct prepared in accordance with the presentinvention. Crossing results in the introduction of a transgene into aplant line by cross pollinating a starting line with a donor plant line.Non-limiting examples of such steps are described in U.S. Pat. No.7,151,204.

Plants may be generated through a process of backcross conversion. Forexample, plants include plants referred to as a backcross convertedgenotype, line, inbred, or hybrid.

Genetic markers may be used to assist in the introgression of one ormore transgenes of the invention from one genetic background intoanother. Marker assisted selection offers advantages relative toconventional breeding in that it can be used to avoid errors caused byphenotypic variations. Further, genetic markers may provide dataregarding the relative degree of elite germplasm in the individualprogeny of a particular cross. For example, when a plant with a desiredtrait which otherwise has a non-agronomically desirable geneticbackground is crossed to an elite parent, genetic markers may be used toselect progeny which not only possess the trait of interest, but alsohave a relatively large proportion of the desired germplasm. In thisway, the number of generations required to introgress one or more traitsinto a particular genetic background is minimized.

The present invention also relates to methods of producing a polypeptideor domain of the present invention comprising (a) cultivating atransgenic plant or a plant cell comprising a polynucleotide encodingthe polypeptide or domain under conditions conducive for production ofthe polypeptide or domain; and optionally (b) recovering the polypeptideor domain.

Removal or Reduction of Catalase Activity

The present invention also relates to methods of producing a mutant of aparent cell, which comprises disrupting or deleting a polynucleotide, ora portion thereof, encoding a polypeptide of the present invention,which results in the mutant cell producing less of the polypeptide thanthe parent cell when cultivated under the same conditions.

The mutant cell may be constructed by reducing or eliminating expressionof the polynucleotide using methods well known in the art, for example,insertions, disruptions, replacements, or deletions. In a preferredaspect, the polynucleotide is inactivated. The polynucleotide to bemodified or inactivated may be, for example, the coding region or a partthereof essential for activity, or a regulatory element required forexpression of the coding region. An example of such a regulatory orcontrol sequence may be a promoter sequence or a functional partthereof, i.e., a part that is sufficient for affecting expression of thepolynucleotide. Other control sequences for possible modificationinclude, but are not limited to, a leader, polyadenylation sequence,propeptide sequence, signal peptide sequence, transcription terminator,and transcriptional activator.

Modification or inactivation of the polynucleotide may be performed bysubjecting the parent cell to mutagenesis and selecting for mutant cellsin which expression of the polynucleotide has been reduced oreliminated. The mutagenesis, which may be specific or random, may beperformed, for example, by use of a suitable physical or chemicalmutagenizing agent, by use of a suitable oligonucleotide, or bysubjecting the DNA sequence to PCR generated mutagenesis. Furthermore,the mutagenesis may be performed by use of any combination of thesemutagenizing agents.

Examples of a physical or chemical mutagenizing agent suitable for thepresent purpose include ultraviolet (UV) irradiation, hydroxylamine,N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), O-methyl hydroxylamine,nitrous acid, ethyl methane sulphonate (EMS), sodium bisulphite, formicacid, and nucleotide analogues.

When such agents are used, the mutagenesis is typically performed byincubating the parent cell to be mutagenized in the presence of themutagenizing agent of choice under suitable conditions, and screeningand/or selecting for mutant cells exhibiting reduced or no expression ofthe gene.

Modification or inactivation of the polynucleotide may also beaccomplished by insertion, substitution, or deletion of one or morenucleotides in the gene or a regulatory element required fortranscription or translation thereof. For example, nucleotides may beinserted or removed so as to result in the introduction of a stop codon,the removal of the start codon, or a change in the open reading frame.Such modification or inactivation may be accomplished by site-directedmutagenesis or PCR generated mutagenesis in accordance with methodsknown in the art. Although, in principle, the modification may beperformed in vivo, i.e., directly on the cell expressing thepolynucleotide to be modified, it is preferred that the modification beperformed in vitro as exemplified below.

An example of a convenient way to eliminate or reduce expression of apolynucleotide is based on techniques of gene replacement, genedeletion, or gene disruption. For example, in the gene disruptionmethod, a nucleic acid sequence corresponding to the endogenouspolynucleotide is mutagenized in vitro to produce a defective nucleicacid sequence that is then transformed into the parent cell to produce adefective gene. By homologous recombination, the defective nucleic acidsequence replaces the endogenous polynucleotide. It may be desirablethat the defective polynucleotide also encodes a marker that may be usedfor selection of transformants in which the polynucleotide has beenmodified or destroyed. In an aspect, the polynucleotide is disruptedwith a selectable marker such as those described herein.

The present invention also relates to methods of inhibiting theexpression of a polypeptide having catalase activity in a cell,comprising administering to the cell or expressing in the cell adouble-stranded RNA (dsRNA) molecule, wherein the dsRNA comprises asubsequence of a polynucleotide of the present invention. In a preferredaspect, the dsRNA is about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 ormore duplex nucleotides in length.

The dsRNA is preferably a small interfering RNA (siRNA) or a micro RNA(miRNA). In a preferred aspect, the dsRNA is small interfering RNA forinhibiting transcription. In another preferred aspect, the dsRNA ismicro RNA for inhibiting translation.

The present invention also relates to such double-stranded RNA (dsRNA)molecules, comprising a portion of the mature polypeptide codingsequence of SEQ ID NO: 7, the mature polypeptide coding sequence of SEQID NO: 1, the mature polypeptide coding sequence of SEQ ID NO: 3, or themature polypeptide coding sequence of SEQ ID NO: 5, for inhibitingexpression of the polypeptide in a cell. While the present invention isnot limited by any particular mechanism of action, the dsRNA can enter acell and cause the degradation of a single-stranded RNA (ssRNA) ofsimilar or identical sequences, including endogenous mRNAs. When a cellis exposed to dsRNA, mRNA from the homologous gene is selectivelydegraded by a process called RNA interference (RNAi).

The dsRNAs of the present invention can be used in gene-silencing. Inone aspect, the invention provides methods to selectively degrade RNAusing a dsRNAi of the present invention. The process may be practiced invitro, ex vivo or in vivo. In one aspect, the dsRNA molecules can beused to generate a loss-of-function mutation in a cell, an organ or ananimal. Methods for making and using dsRNA molecules to selectivelydegrade RNA are well known in the art; see, for example, U.S. Pat. Nos.6,489,127; 6,506,559; 6,511,824; and 6,515,109.

The present invention further relates to a mutant cell of a parent cellthat comprises a disruption or deletion of a polynucleotide encoding thepolypeptide or a control sequence thereof or a silenced gene encodingthe polypeptide, which results in the mutant cell producing less of thepolypeptide or no polypeptide compared to the parent cell.

The polypeptide-deficient mutant cells are particularly useful as hostcells for expression of native and heterologous polypeptides. Therefore,the present invention further relates to methods of producing a nativeor heterologous polypeptide, comprising (a) cultivating the mutant cellunder conditions conducive for production of the polypeptide; andoptionally (b) recovering the polypeptide. The term “heterologouspolypeptides” means polypeptides that are not native to the host cell,e.g., a variant of a native protein. The host cell may comprise morethan one copy of a polynucleotide encoding the native or heterologouspolypeptide.

The methods used for cultivation and purification of the product ofinterest may be performed by methods known in the art.

The methods of the present invention for producing an essentiallycatalase-free product are of particular interest in the production ofeukaryotic polypeptides, in particular fungal proteins such as enzymes.The catalase-deficient cells may also be used to express heterologousproteins of pharmaceutical interest such as hormones, growth factors,receptors, and the like. The term “eukaryotic polypeptides” includes notonly native polypeptides, but also those polypeptides, e.g., enzymes,which have been modified by amino acid substitutions, deletions oradditions, or other such modifications to enhance activity,thermostability, pH tolerance and the like.

In a further aspect, the present invention relates to a protein productessentially free from catalase activity that is produced by a method ofthe present invention.

Fermentation Broth Formulations or Cell Compositions

The present invention also relates to a fermentation broth formulationor a cell composition comprising a polypeptide of the present invention.The fermentation broth product further comprises additional ingredientsused in the fermentation process, such as, for example, cells(including, the host cells containing the gene encoding the polypeptideof the present invention which are used to produce the polypeptide ofinterest), cell debris, biomass, fermentation media and/or fermentationproducts. In some embodiments, the composition is a cell-killed wholebroth containing organic acid(s), killed cells and/or cell debris, andculture medium.

The term “fermentation broth” as used herein refers to a preparationproduced by cellular fermentation that undergoes no or minimal recoveryand/or purification. For example, fermentation broths are produced whenmicrobial cultures are grown to saturation, incubated undercarbon-limiting conditions to allow protein synthesis (e.g., expressionof enzymes by host cells) and secretion into cell culture medium. Thefermentation broth can contain unfractionated or fractionated contentsof the fermentation materials derived at the end of the fermentation.Typically, the fermentation broth is unfractionated and comprises thespent culture medium and cell debris present after the microbial cells(e.g., filamentous fungal cells) are removed, e.g., by centrifugation.In some embodiments, the fermentation broth contains spent cell culturemedium, extracellular enzymes, and viable and/or nonviable microbialcells.

In an embodiment, the fermentation broth formulation and cellcompositions comprise a first organic acid component comprising at leastone 1-5 carbon organic acid and/or a salt thereof and a second organicacid component comprising at least one 6 or more carbon organic acidand/or a salt thereof. In a specific embodiment, the first organic acidcomponent is acetic acid, formic acid, propionic acid, a salt thereof,or a mixture of two or more of the foregoing and the second organic acidcomponent is benzoic acid, cyclohexanecarboxylic acid, 4-methylvalericacid, phenylacetic acid, a salt thereof, or a mixture of two or more ofthe foregoing.

In one aspect, the composition contains an organic acid(s), andoptionally further contains killed cells and/or cell debris. In oneembodiment, the killed cells and/or cell debris are removed from acell-killed whole broth to provide a composition that is free of thesecomponents.

The fermentation broth formulations or cell compositions may furthercomprise a preservative and/or anti-microbial (e.g., bacteriostatic)agent, including, but not limited to, sorbitol, sodium chloride,potassium sorbate, and others known in the art.

The fermentation broth formulations or cell compositions may furthercomprise multiple enzymatic activities, such as one or more (e.g.,several) enzymes selected from the group consisting of a cellulase, aGH61 polypeptide having cellulolytic enhancing activity, ahemicellulase, an esterase, an expansin, a laccase, a ligninolyticenzyme, a pectinase, a peroxidase, a protease, and a swollenin. Thefermentation broth formulations or cell compositions may also compriseone or more (e.g., several) enzymes selected from the group consistingof a hydrolase, an isomerase, a ligase, a lyase, an oxidoreductase, or atransferase, e.g., an alpha-galactosidase, alpha-glucosidase,aminopeptidase, amylase, beta-galactosidase, beta-glucosidase,beta-xylosidase, carbohydrase, carboxypeptidase, catalase,cellobiohydrolase, cellulase, chitinase, cutinase, cyclodextringlycosyltransferase, deoxyribonuclease, endoglucanase, esterase,glucoamylase, invertase, laccase, lipase, mannosidase, mutanase,oxidase, pectinolytic enzyme, peroxidase, phytase, polyphenoloxidase,proteolytic enzyme, ribonuclease, transglutaminase, or xylanase.

The cell-killed whole broth or composition may contain theunfractionated contents of the fermentation materials derived at the endof the fermentation. Typically, the cell-killed whole broth orcomposition contains the spent culture medium and cell debris presentafter the microbial cells (e.g., filamentous fungal cells) are grown tosaturation, incubated under carbon-limiting conditions to allow proteinsynthesis (e.g., expression of cellulase and/or glucosidase enzyme(s)).In some embodiments, the cell-killed whole broth or composition containsthe spent cell culture medium, extracellular enzymes, and killedfilamentous fungal cells. In some embodiments, the microbial cellspresent in the cell-killed whole broth or composition can bepermeabilized and/or lysed using methods known in the art.

A whole broth or cell composition as described herein is typically aliquid, but may contain insoluble components, such as killed cells, celldebris, culture media components, and/or insoluble enzyme(s). In someembodiments, insoluble components may be removed to provide a clarifiedliquid composition.

The whole broth formulations and cell compositions of the presentinvention may be produced by a method described in WO 90/15861 or WO2010/096673.

Examples are given below of preferred uses of the compositions of thepresent invention. The dosage of the composition and other conditionsunder which the composition is used may be determined on the basis ofmethods known in the art.

Enzyme Compositions

The present invention also relates to compositions comprising apolypeptide of the present invention. Preferably, the compositions areenriched in such a polypeptide. The term “enriched” indicates that thecatalase activity of the composition has been increased, e.g., with anenrichment factor of at least 1.1.

The compositions may comprise a polypeptide of the present invention asthe major enzymatic component, e.g., a mono-component composition.Alternatively, the compositions may comprise multiple enzymaticactivities, such as one or more (e.g., several) enzymes selected fromthe group consisting of a cellulase, a GH61 polypeptide havingcellulolytic enhancing activity, a hemicellulase, an esterase, anexpansin, a laccase, a ligninolytic enzyme, a pectinase, a peroxidase, aprotease, and a swollenin. The compositions may also comprise one ormore (e.g., several) enzymes selected from the group consisting of ahydrolase, an isomerase, a ligase, a lyase, an oxidoreductase, or atransferase, e.g., an alpha-galactosidase, alpha-glucosidase,aminopeptidase, amylase, beta-galactosidase, beta-glucosidase,beta-xylosidase, carbohydrase, carboxypeptidase, catalase,cellobiohydrolase, cellulase, chitinase, cutinase, cyclodextringlycosyltransferase, deoxyribonuclease, endoglucanase, esterase,glucoamylase, invertase, laccase, lipase, mannosidase, mutanase,oxidase, pectinolytic enzyme, peroxidase, phytase, polyphenoloxidase,proteolytic enzyme, ribonuclease, transglutaminase, or xylanase. Thecompositions may be prepared in accordance with methods known in the artand may be in the form of a liquid or a dry composition. Thecompositions may be stabilized in accordance with methods known in theart.

Examples are given below of preferred uses of the compositions of thepresent invention. The dosage of the composition and other conditionsunder which the composition is used may be determined on the basis ofmethods known in the art.

Uses

In general terms, the polypeptide can be used in any situation in whichit is desired to remove residual hydrogen peroxide from a mixture towhich hydrogen peroxide has been added or generated, e.g., forpasteurization or bleaching.

The polypeptides having catalase activity of the present invention canbe used commercially for diagnostic enzyme kits, for the enzymaticproduction of sodium gluconate from glucose, for the neutralization ofH₂O₂ waste, and for the removal of H₂O₂ and/or generation of O₂ in foodsand beverages using methods well established in the art.

In one aspect, the present invention also relates to methods forremoving hydrogen peroxide, comprising treating a mixture to whichhydrogen peroxide has been added or generated with a polypeptide of thepresent invention.

In one aspect, the present invention relates to a method for removinghydrogen peroxide from textile.

During textile manufacturing, hydrogen peroxide is used in the bleachingstep to completely remove colored impurities, improve absorbency, andachieve adequate whiteness and dyeability. However, excess peroxideproduct remains on the textile and it can interfere with and have anadverse affect on subsequent dyeings with anionic dyes, for example,reactive dyes where the dye is in part or totally destroyed. Genenrally,catalase is applied after the bleaching step to help to destroy excesshydrogen peroxide. In another aspect, the present invention also relatesto methods for generating molecular oxygen, comprising treating amixture to which hydrogen peroxide has been added or generated with apolypeptide of the present invention.

The present invention is also directed to the following processes forusing the polypeptides having catalase activity, or compositionsthereof.

The present invention also relates to processes for degrading orconverting a cellulosic material, comprising: treating the cellulosicmaterial with an enzyme composition in the presence of a polypeptidehaving catalase activity of the present invention. In one aspect, theprocesses further comprise recovering the degraded or convertedcellulosic material. Soluble products of degradation or conversion ofthe cellulosic material can be separated from insoluble cellulosicmaterial using a method known in the art such as, for example,centrifugation, filtration, or gravity settling.

The present invention also relates to processes of producing afermentation product, comprising: (a) saccharifying a cellulosicmaterial with an enzyme composition in the presence of a polypeptidehaving catalase activity of the present invention; (b) fermenting thesaccharified cellulosic material with one or more (e.g., several)fermenting microorganisms to produce the fermentation product; and (c)recovering the fermentation product from the fermentation. The presentinvention also relates to processes of fermenting a cellulosic material,comprising: fermenting the cellulosic material with one or more (e.g.,several) fermenting microorganisms, wherein the cellulosic material issaccharified with an enzyme composition in the presence of a polypeptidehaving catalase activity of the present invention. In one aspect, thefermenting of the cellulosic material produces a fermentation product.In another aspect, the processes further comprise recovering thefermentation product from the fermentation.

The processes of the present invention can be used to saccharify thecellulosic material to fermentable sugars and to convert the fermentablesugars to many useful fermentation products, e.g., fuel, potableethanol, and/or platform chemicals (e.g., acids, alcohols, ketones,gases, and the like). The production of a desired fermentation productfrom the cellulosic material typically involves pretreatment, enzymatichydrolysis (saccharification), and fermentation.

The processing of the cellulosic material according to the presentinvention can be accomplished using methods conventional in the art.Moreover, the processes of the present invention can be implementedusing any conventional biomass processing apparatus configured tooperate in accordance with the invention.

Hydrolysis (saccharification) and fermentation, separate orsimultaneous, include, but are not limited to, separate hydrolysis andfermentation (SHF); simultaneous saccharification and fermentation(SSF); simultaneous saccharification and co-fermentation (SSCF); hybridhydrolysis and fermentation (HHF); separate hydrolysis andco-fermentation (SHCF); hybrid hydrolysis and co-fermentation (HHCF);and direct microbial conversion (DMC), also sometimes calledconsolidated bioprocessing (CBP). SHF uses separate process steps tofirst enzymatically hydrolyze the cellulosic material to fermentablesugars, e.g., glucose, cellobiose, and pentose monomers, and thenferment the fermentable sugars to ethanol. In SSF, the enzymatichydrolysis of the cellulosic material and the fermentation of sugars toethanol are combined in one step (Philippidis, G. P., 1996, Cellulosebioconversion technology, in Handbook on Bioethanol: Production andUtilization, Wyman, C. E., ed., Taylor & Francis, Washington, D.C.,179-212). SSCF involves the co-fermentation of multiple sugars (Sheehan,J., and Himmel, M., 1999, Enzymes, energy and the environment: Astrategic perspective on the U.S. Department of Energy's research anddevelopment activities for bioethanol, Biotechnol. Prog. 15: 817-827).HHF involves a separate hydrolysis step, and in addition a simultaneoussaccharification and hydrolysis step, which can be carried out in thesame reactor. The steps in an HHF process can be carried out atdifferent temperatures, i.e., high temperature enzymaticsaccharification followed by SSF at a lower temperature that thefermentation strain can tolerate. DMC combines all three processes(enzyme production, hydrolysis, and fermentation) in one or more (e.g.,several) steps where the same organism is used to produce the enzymesfor conversion of the cellulosic material to fermentable sugars and toconvert the fermentable sugars into a final product (Lynd, L. R.,Weimer, P. J., van Zyl, W. H., and Pretorius, I. S., 2002, Microbialcellulose utilization: Fundamentals and biotechnology, Microbiol. Mol.Biol. Reviews 66: 506-577). It is understood herein that any methodknown in the art comprising pretreatment, enzymatic hydrolysis(saccharification), fermentation, or a combination thereof, can be usedin the practicing the processes of the present invention.

A conventional apparatus can include a fed-batch stirred reactor, abatch stirred reactor, a continuous flow stirred reactor withultrafiltration, and/or a continuous plug-flow column reactor (Fernandade Castilhos Corazza, Flávio Faria de Moraes, Gisella Maria Zanin andIvo Neitzel, 2003, Optimal control in fed-batch reactor for thecellobiose hydrolysis, Acta Scientiarum. Technology 25: 33-38; Gusakov,A. V., and Sinitsyn, A. P., 1985, Kinetics of the enzymatic hydrolysisof cellulose: 1. A mathematical model for a batch reactor process, Enz.Microb. Technol. 7: 346-352), an attrition reactor (Ryu, S. K., and Lee,J. M., 1983, Bioconversion of waste cellulose by using an attritionbioreactor, Biotechnol. Bioeng. 25: 53-65), or a reactor with intensivestirring induced by an electromagnetic field (Gusakov, A. V., Sinitsyn,A. P., Davydkin, I. Y., Davydkin, V. Y., Protas, O. V., 1996,Enhancement of enzymatic cellulose hydrolysis using a novel type ofbioreactor with intensive stirring induced by electromagnetic field,Appl. Biochem. Biotechnol. 56: 141-153). Additional reactor typesinclude fluidized bed, upflow blanket, immobilized, and extruder typereactors for hydrolysis and/or fermentation.

Pretreatment. In practicing the processes of the present invention, anypretreatment process known in the art can be used to disrupt plant cellwall components of the cellulosic material (Chandra et al, 2007,Substrate pretreatment: The key to effective enzymatic hydrolysis oflignocellulosics?, Adv. Biochem. Engin./Biotechnol. 108: 67-93; Galbeand Zacchi, 2007, Pretreatment of lignocellulosic materials forefficient bioethanol production, Adv. Biochem. Engin./Biotechnol. 108:41-65; Hendriks and Zeeman, 2009, Pretreatments to enhance thedigestibility of lignocellulosic biomass, Bioresource Technol. 100:10-18; Mosier et al., 2005, Features of promising technologies forpretreatment of lignocellulosic biomass, Bioresource Technol. 96:673-686; Taherzadeh and Karimi, 2008, Pretreatment of lignocellulosicwastes to improve ethanol and biogas production: A review, Int. J. ofMol. Sci. 9: 1621-1651; Yang and Wyman, 2008, Pretreatment: the key tounlocking low-cost cellulosic ethanol, Biofuels Bioproducts andBiorefining-Biofpr. 2: 26-40).

The cellulosic material can also be subjected to particle sizereduction, sieving, pre-soaking, wetting, washing, and/or conditioningprior to pretreatment using methods known in the art.

Conventional pretreatments include, but are not limited to, steampretreatment (with or without explosion), dilute acid pretreatment, hotwater pretreatment, alkaline pretreatment, lime pretreatment, wetoxidation, wet explosion, ammonia fiber explosion, organosolvpretreatment, and biological pretreatment. Additional pretreatmentsinclude ammonia percolation, ultrasound, electroporation, microwave,supercritical CO₂, supercritical H₂O, ozone, ionic liquid, and gammairradiation pretreatments.

The cellulosic material can be pretreated before hydrolysis and/orfermentation. Pretreatment is preferably performed prior to thehydrolysis. Alternatively, the pretreatment can be carried outsimultaneously with enzyme hydrolysis to release fermentable sugars,such as glucose, xylose, and/or cellobiose. In most cases thepretreatment step itself results in some conversion of biomass tofermentable sugars (even in absence of enzymes).

Steam Pretreatment. In steam pretreatment, the cellulosic material isheated to disrupt the plant cell wall components, including lignin,hemicellulose, and cellulose to make the cellulose and other fractions,e.g., hemicellulose, accessible to enzymes. The cellulosic material ispassed to or through a reaction vessel where steam is injected toincrease the temperature to the required temperature and pressure and isretained therein for the desired reaction time. Steam pretreatment ispreferably performed at 140-250° C., e.g., 160-200° C. or 170-190° C.,where the optimal temperature range depends on addition of a chemicalcatalyst. Residence time for the steam pretreatment is preferably 1-60minutes, e.g., 1-30 minutes, 1-20 minutes, 3-12 minutes, or 4-10minutes, where the optimal residence time depends on temperature rangeand addition of a chemical catalyst. Steam pretreatment allows forrelatively high solids loadings, so that the cellulosic material isgenerally only moist during the pretreatment. The steam pretreatment isoften combined with an explosive discharge of the material after thepretreatment, which is known as steam explosion, that is, rapid flashingto atmospheric pressure and turbulent flow of the material to increasethe accessible surface area by fragmentation (Duff and Murray, 1996,Bioresource Technology 855: 1-33; Galbe and Zacchi, 2002, Appl.Microbiol. Biotechnol. 59: 618-628; U.S. Patent Application No.20020164730). During steam pretreatment, hemicellulose acetyl groups arecleaved and the resulting acid autocatalyzes partial hydrolysis of thehemicellulose to monosaccharides and oligosaccharides. Lignin is removedto only a limited extent.

Chemical Pretreatment: The term “chemical treatment” refers to anychemical pretreatment that promotes the separation and/or release ofcellulose, hemicellulose, and/or lignin. Such a pretreatment can convertcrystalline cellulose to amorphous cellulose. Examples of suitablechemical pretreatment processes include, for example, dilute acidpretreatment, lime pretreatment, wet oxidation, ammonia fiber/freezeexplosion (AFEX), ammonia percolation (APR), ionic liquid, andorganosolv pretreatments.

A catalyst such as H₂SO₄ or SO₂ (typically 0.3 to 5% w/w) is often addedprior to steam pretreatment, which decreases the time and temperature,increases the recovery, and improves enzymatic hydrolysis (Ballesteroset al, 2006, Appl. Biochem. Biotechnol. 129-132: 496-508; Varga et al.,2004, Appl. Biochem. Biotechnol. 113-116: 509-523; Sassner et al, 2006,Enzyme Microb. Technol. 39: 756-762). In dilute acid pretreatment, thecellulosic material is mixed with dilute acid, typically H₂SO₄, andwater to form a slurry, heated by steam to the desired temperature, andafter a residence time flashed to atmospheric pressure. The dilute acidpretreatment can be performed with a number of reactor designs, e.g.,plug-flow reactors, counter-current reactors, or continuouscounter-current shrinking bed reactors (Duff and Murray, 1996, supra;Schell et al, 2004, Bioresource Technol. 91: 179-188; Lee et al, 1999,Adv. Biochem. Eng. Biotechnol. 65: 93-115).

Several methods of pretreatment under alkaline conditions can also beused. These alkaline pretreatments include, but are not limited to,sodium hydroxide, lime, wet oxidation, ammonia percolation (APR), andammonia fiber/freeze explosion (AFEX).

Lime pretreatment is performed with calcium oxide or calcium hydroxideat temperatures of 85-150° C. and residence times from 1 hour to severaldays (Wyman et al, 2005, Bioresource Technol 96: 1959-1966; Mosier etal., 2005, Bioresource Technol 96: 673-686). WO 2006/110891, WO2006/110899, WO 2006/110900, and WO 2006/110901 disclose pretreatmentmethods using ammonia.

Wet oxidation is a thermal pretreatment performed typically at 180-200°C. for 5-15 minutes with addition of an oxidative agent such as hydrogenperoxide or over-pressure of oxygen (Schmidt and Thomsen, 1998,Bioresource Technol 64: 139-151; Palonen et al, 2004, Appl. Biochem.Biotechnol. 117: 1-17; Varga et al, 2004, Biotechnol. Bioeng. 88:567-574; Martin et al., 2006, J. Chem. Technol. Biotechnol. 81:1669-1677). The pretreatment is performed preferably at 1-40% drymatter, e.g., 2-30% dry matter or 5-20% dry matter, and often theinitial pH is increased by the addition of alkali such as sodiumcarbonate.

A modification of the wet oxidation pretreatment method, known as wetexplosion (combination of wet oxidation and steam explosion) can handledry matter up to 30%. In wet explosion, the oxidizing agent isintroduced during pretreatment after a certain residence time. Thepretreatment is then ended by flashing to atmospheric pressure (WO2006/032282).

Ammonia fiber explosion (AFEX) involves treating the cellulosic materialwith liquid or gaseous ammonia at moderate temperatures such as 90-150°C. and high pressure such as 17-20 bar for 5-10 minutes, where the drymatter content can be as high as 60% (Gollapalli et al., 2002, Appl.Biochem. Biotechnol. 98: 23-35; Chundawat et al., 2007, Biotechnol.Bioeng. 96: 219-231; Alizadeh et al., 2005, Appl. Biochem. Biotechnol121: 1133-1141; Teymouri et al., 2005, Bioresource Technol. 96:2014-2018). During AFEX pretreatment cellulose and hemicelluloses remainrelatively intact. Lignin-carbohydrate complexes are cleaved.

Organosolv pretreatment delignifies the cellulosic material byextraction using aqueous ethanol (40-60% ethanol) at 160-200° C. for30-60 minutes (Pan et al., 2005, Biotechnol. Bioeng. 90: 473-481; Pan etal., 2006, Biotechnol. Bioeng. 94: 851-861; Kurabi et al., 2005, Appl.Biochem. Biotechnol. 121: 219-230). Sulphuric acid is usually added as acatalyst. In organosolv pretreatment, the majority of hemicellulose andlignin is removed.

Other examples of suitable pretreatment methods are described by Schellet al., 2003, Appl. Biochem. and Biotechnol. Vol. 105-108, p. 69-85, andMosier et al., 2005, Bioresource Technology 96: 673-686, and U.S.Published Application 2002/0164730.

In one aspect, the chemical pretreatment is preferably carried out as adilute acid treatment, and more preferably as a continuous dilute acidtreatment. The acid is typically sulfuric acid, but other acids can alsobe used, such as acetic acid, citric acid, nitric acid, phosphoric acid,tartaric acid, succinic acid, hydrogen chloride, or mixtures thereof.Mild acid treatment is conducted in the pH range of preferably 1-5,e.g., 1-4 or 1-2.5. In one aspect, the acid concentration is in therange from preferably 0.01 to 10 wt % acid, e.g., 0.05 to 5 wt % acid or0.1 to 2 wt % acid. The acid is contacted with the cellulosic materialand held at a temperature in the range of preferably 140-200° C., e.g.,165-190° C., for periods ranging from 1 to 60 minutes.

In another aspect, pretreatment takes place in an aqueous slurry. Inpreferred aspects, the cellulosic material is present duringpretreatment in amounts preferably between 10-80 wt %, e.g., 20-70 wt %or 30-60 wt %, such as around 40 wt %. The pretreated cellulosicmaterial can be unwashed or washed using any method known in the art,e.g., washed with water.

Mechanical Pretreatment or Physical Pretreatment: The term “mechanicalpretreatment” or “physical pretreatment” refers to any pretreatment thatpromotes size reduction of particles. For example, such pretreatment caninvolve various types of grinding or milling (e.g., dry milling, wetmilling, or vibratory ball milling).

The cellulosic material can be pretreated both physically (mechanically)and chemically. Mechanical or physical pretreatment can be coupled withsteaming/steam explosion, hydrothermolysis, dilute or mild acidtreatment, high temperature, high pressure treatment, irradiation (e.g.,microwave irradiation), or combinations thereof. In one aspect, highpressure means pressure in the range of preferably about 100 to about400 psi, e.g., about 150 to about 250 psi. In another aspect, hightemperature means temperatures in the range of about 100 to about 300°C., e.g., about 140 to about 200° C. In a preferred aspect, mechanicalor physical pretreatment is performed in a batch-process using a steamgun hydrolyzer system that uses high pressure and high temperature asdefined above, e.g., a Sunds Hydrolyzer available from Sunds DefibratorAB, Sweden. The physical and chemical pretreatments can be carried outsequentially or simultaneously, as desired.

Accordingly, in a preferred aspect, the cellulosic material is subjectedto physical (mechanical) or chemical pretreatment, or any combinationthereof, to promote the separation and/or release of cellulose,hemicellulose, and/or lignin.

Biological Pretreatment: The term “biological pretreatment” refers toany biological pretreatment that promotes the separation and/or releaseof cellulose, hemicellulose, and/or lignin from the cellulosic material.Biological pretreatment techniques can involve applyinglignin-solubilizing microorganisms and/or enzymes (see, for example,Hsu, T.-A., 1996, Pretreatment of biomass, in Handbook on Bioethanol:Production and Utilization, Wyman, C. E., ed., Taylor & Francis,Washington, D.C., 179-212; Ghosh and Singh, 1993, Physicochemical andbiological treatments for enzymatic/microbial conversion of cellulosicbiomass, Adv. Appl. Microbiol. 39: 295-333; McMillan, J. D., 1994,Pretreating lignocellulosic biomass: a review, in Enzymatic Conversionof Biomass for Fuels Production, Himmel, M. E., Baker, J. O., andOverend, R. P., eds., ACS Symposium Series 566, American ChemicalSociety, Washington, D.C., chapter 15; Gong, C. S., Cao, N. J., Du, J.,and Tsao, G. T., 1999, Ethanol production from renewable resources, inAdvances in Biochemical Engineering/Biotechnology, Scheper, T., ed.,Springer-Verlag Berlin Heidelberg, Germany, 65: 207-241; Olsson andHahn-Hagerdal, 1996, Fermentation of lignocellulosic hydrolysates forethanol production, Enz. Microb. Tech. 18: 312-331; and Vallander andEriksson, 1990, Production of ethanol from lignocellulosic materials:State of the art, Adv. Biochem. Eng./Biotechnol. 42: 63-95).

Saccharification. In the hydrolysis step, also known assaccharification, the cellulosic material, e.g., pretreated, ishydrolyzed to break down cellulose and/or hemicellulose to fermentablesugars, such as glucose, cellobiose, xylose, xylulose, arabinose,mannose, galactose, and/or soluble oligosaccharides. The hydrolysis isperformed enzymatically by an enzyme composition as described herein inthe presence of a polypeptide having catalase activity of the presentinvention. The enzyme components of the compositions can be addedsimultaneously or sequentially.

Enzymatic hydrolysis is preferably carried out in a suitable aqueousenvironment under conditions that can be readily determined by oneskilled in the art. In one aspect, hydrolysis is performed underconditions suitable for the activity of the enzyme components, i.e.,optimal for the enzyme components. The hydrolysis can be carried out asa fed batch or continuous process where the cellulosic material is fedgradually to, for example, an enzyme containing hydrolysis solution.

The saccharification is generally performed in stirred-tank reactors orfermentors under controlled pH, temperature, and mixing conditions.Suitable process time, temperature and pH conditions can readily bedetermined by one skilled in the art. For example, the saccharificationcan last up to 200 hours, but is typically performed for preferablyabout 12 to about 120 hours, e.g., about 16 to about 72 hours or about24 to about 48 hours. The temperature is in the range of preferablyabout 25° C. to about 70° C., e.g., about 30° C. to about 65° C., about40° C. to about 60° C., or about 50° C. to about 55° C. The pH is in therange of preferably about 3 to about 8, e.g., about 3.5 to about 7,about 4 to about 6, or about 5.0 to about 5.5. The dry solids content isin the range of preferably about 5 to about 50 wt %, e.g., about 10 toabout 40 wt % or about 20 to about 30 wt %.

The enzyme compositions can comprise any protein useful in degrading orconverting the cellulosic material.

In one aspect, the enzyme composition comprises or further comprises oneor more (e.g., several) proteins/polypeptides selected from the groupconsisting of a cellulase, a GH61 polypeptide having cellulolyticenhancing activity, a hemicellulase, an esterase, an expansin, alaccase, a ligninolytic enzyme, a pectinase, a peroxidase, a protease,and a swollenin. In another aspect, the cellulase is preferably one ormore (e.g., several) enzymes selected from the group consisting of anendoglucanase, a cellobiohydrolase, and a beta-glucosidase. In anotheraspect, the hemicellulase is preferably one or more (e.g., several)enzymes selected from the group consisting of an acetylmannan esterase,an acetylxylan esterase, an arabinanase, an arabinofuranosidase, acoumaric acid esterase, a feruloyl esterase, a galactosidase, aglucuronidase, a glucuronoyl esterase, a mannanase, a mannosidase, axylanase, and a xylosidase.

In another aspect, the enzyme composition comprises one or more (e.g.,several) cellulolytic enzymes. In another aspect, the enzyme compositioncomprises or further comprises one or more (e.g., several)hemicellulolytic enzymes. In another aspect, the enzyme compositioncomprises one or more (e.g., several) cellulolytic enzymes and one ormore (e.g., several) hemicellulolytic enzymes. In another aspect, theenzyme composition comprises one or more (e.g., several) enzymesselected from the group of cellulolytic enzymes and hemicellulolyticenzymes. In another aspect, the enzyme composition comprises anendoglucanase. In another aspect, the enzyme composition comprises acellobiohydrolase. In another aspect, the enzyme composition comprises abeta-glucosidase. In another aspect, the enzyme composition comprises apolypeptide having cellulolytic enhancing activity. In another aspect,the enzyme composition comprises an endoglucanase and a polypeptidehaving cellulolytic enhancing activity. In another aspect, the enzymecomposition comprises a cellobiohydrolase and a polypeptide havingcellulolytic enhancing activity. In another aspect, the enzymecomposition comprises a beta-glucosidase and a polypeptide havingcellulolytic enhancing activity. In another aspect, the enzymecomposition comprises an endoglucanase and a cellobiohydrolase. Inanother aspect, the enzyme composition comprises an endoglucanase and abeta-glucosidase. In another aspect, the enzyme composition comprises acellobiohydrolase and a beta-glucosidase. In another aspect, the enzymecomposition comprises an endoglucanase, a cellobiohydrolase, and apolypeptide having cellulolytic enhancing activity. In another aspect,the enzyme composition comprises an endoglucanase, a beta-glucosidase,and a polypeptide having cellulolytic enhancing activity. In anotheraspect, the enzyme composition comprises a cellobiohydrolase, abeta-glucosidase, and a polypeptide having cellulolytic enhancingactivity. In another aspect, the enzyme composition comprises anendoglucanase, a cellobiohydrolase, and a beta-glucosidase. In anotheraspect, the enzyme composition comprises an endoglucanase, acellobiohydrolase, a beta-glucosidase, and a polypeptide havingcellulolytic enhancing activity.

In another aspect, the enzyme composition comprises an acetylmannanesterase. In another aspect, the enzyme composition comprises anacetylxylan esterase. In another aspect, the enzyme compositioncomprises an arabinanase (e.g., alpha-L-arabinanase). In another aspect,the enzyme composition comprises an arabinofuranosidase (e.g.,alpha-L-arabinofuranosidase). In another aspect, the enzyme compositioncomprises a coumaric acid esterase. In another aspect, the enzymecomposition comprises a feruloyl esterase. In another aspect, the enzymecomposition comprises a galactosidase (e.g., alpha-galactosidase and/orbeta-galactosidase). In another aspect, the enzyme composition comprisesa glucuronidase (e.g., alpha-D-glucuronidase). In another aspect, theenzyme composition comprises a glucuronoyl esterase. In another aspect,the enzyme composition comprises a mannanase. In another aspect, theenzyme composition comprises a mannosidase (e.g., beta-mannosidase). Inanother aspect, the enzyme composition comprises a xylanase. In apreferred aspect, the xylanase is a Family 10 xylanase. In anotheraspect, the enzyme composition comprises a xylosidase (e.g.,beta-xylosidase).

In another aspect, the enzyme composition comprises an esterase. Inanother aspect, the enzyme composition comprises an expansin. In anotheraspect, the enzyme composition comprises a laccase. In another aspect,the enzyme composition comprises a ligninolytic enzyme. In a preferredaspect, the ligninolytic enzyme is a manganese peroxidase. In anotherpreferred aspect, the ligninolytic enzyme is a lignin peroxidase. Inanother preferred aspect, the ligninolytic enzyme is a H₂O₂-producingenzyme. In another aspect, the enzyme composition comprises a pectinase.In another aspect, the enzyme composition comprises a peroxidase. Inanother aspect, the enzyme composition comprises a protease. In anotheraspect, the enzyme composition comprises a swollenin

In the processes of the present invention, the enzyme(s) can be addedprior to or during saccharification, saccharification and fermentation,or fermentation.

One or more (e.g., several) components of the enzyme composition may bewild-type proteins, recombinant proteins, or a combination of wild-typeproteins and recombinant proteins. For example, one or more (e.g.,several) components may be native proteins of a cell, which is used as ahost cell to express recombinantly one or more (e.g., several) othercomponents of the enzyme composition. One or more (e.g., several)components of the enzyme composition may be produced as monocomponents,which are then combined to form the enzyme composition. The enzymecomposition may be a combination of multicomponent and monocomponentprotein preparations.

The enzymes used in the processes of the present invention may be in anyform suitable for use, such as, for example, a fermentation brothformulationor a cell composition, a cell lysate with or without cellulardebris, a semi-purified or purified enzyme preparation, or a host cellas a source of the enzymes. The enzyme composition may be a dry powderor granulate, a non-dusting granulate, a liquid, a stabilized liquid, ora stabilized protected enzyme. Liquid enzyme preparations may, forinstance, be stabilized by adding stabilizers such as a sugar, a sugaralcohol or another polyol, and/or lactic acid or another organic acidaccording to established processes.

The optimum amounts of the enzymes and polypeptides having catalaseactivity depend on several factors including, but not limited to, themixture of cellulolytic and/or hemicellulolytic enzyme components, thecellulosic material, the concentration of cellulosic material, thepretreatment(s) of the cellulosic material, temperature, time, pH, andinclusion of fermenting organism (e.g., yeast for SimultaneousSaccharification and Fermentation).

In one aspect, an effective amount of cellulolytic or hemicellulolyticenzyme to the cellulosic material is about 0.5 to about 50 mg, e.g.,about 0.5 to about 40 mg, about 0.5 to about 25 mg, about 0.75 to about20 mg, about 0.75 to about 15 mg, about 0.5 to about 10 mg, or about 2.5to about 10 mg per g of the cellulosic material.

In another aspect, an effective amount of a polypeptide having catalaseactivity to the cellulosic material is about 0.01 to about 50.0 mg,e.g., about 0.01 to about 40 mg, about 0.01 to about 30 mg, about 0.01to about 20 mg, about 0.01 to about 10 mg, about 0.01 to about 5 mg,about 0.025 to about 1.5 mg, about 0.05 to about 1.25 mg, about 0.075 toabout 1.25 mg, about 0.1 to about 1.25 mg, about 0.15 to about 1.25 mg,or about 0.25 to about 1.0 mg per g of the cellulosic material.

In another aspect, an effective amount of a polypeptide having catalaseactivity to cellulolytic or hemicellulolytic enzyme is about 0.005 toabout 1.0 g, e.g., about 0.01 to about 1.0 g, about 0.15 to about 0.75g, about 0.15 to about 0.5 g, about 0.1 to about 0.5 g, about 0.1 toabout 0.25 g, or about 0.05 to about 0.2 g per g of cellulolytic orhemicellulolytic enzyme.

The polypeptides having cellulolytic enzyme activity or hemicellulolyticenzyme activity as well as other proteins/polypeptides useful in thedegradation of the cellulosic material, e.g., GH61 polypeptides havingcellulolytic enhancing activity (collectively hereinafter “polypeptideshaving enzyme activity”) can be derived or obtained from any suitableorigin, including, bacterial, fungal, yeast, plant, or mammalian origin.The term “obtained” also means herein that the enzyme may have beenproduced recombinantly in a host organism employing methods describedherein, wherein the recombinantly produced enzyme is either native orforeign to the host organism or has a modified amino acid sequence,e.g., having one or more (e.g., several) amino acids that are deleted,inserted and/or substituted, i.e., a recombinantly produced enzyme thatis a mutant and/or a fragment of a native amino acid sequence or anenzyme produced by nucleic acid shuffling processes known in the art.Encompassed within the meaning of a native enzyme are natural variantsand within the meaning of a foreign enzyme are variants obtainedrecombinantly, such as by site-directed mutagenesis or shuffling.

A polypeptide having enzyme activity may be a bacterial polypeptide. Forexample, the polypeptide may be a Gram-positive bacterial polypeptidesuch as a Bacillus, Streptococcus, Streptomyces, Staphylococcus,Enterococcus, Lactobacillus, Lactococcus, Clostridium, Geobacillus,Caldicellulosituptor, Acidothermus, Thermobifidia, or Oceanobacilluspolypeptide having enzyme activity, or a Gram negative bacterialpolypeptide such as an E. coli, Pseudomonas, Salmonella, Campylobacter,Helicobacter, Flavobacterium, Fusobacterium, Ilyobacter, Neisseria, orUreaplasma polypeptide having enzyme activity.

In one aspect, the polypeptide is a Bacillus alkalophilus, Bacillusamyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillusclausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacilluslentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus,Bacillus stearothermophilus, Bacillus subtilis, or Bacillusthuringiensis polypeptide having enzyme activity.

In another aspect, the polypeptide is a Streptococcus equisimilis,Streptococcus pyogenes, Streptococcus uberis, or Streptococcus equisubsp. Zooepidemicus polypeptide having enzyme activity.

In another aspect, the polypeptide is a Streptomyces achromogenes,Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus,or Streptomyces lividans polypeptide having enzyme activity.

The polypeptide having enzyme activity may also be a fungal polypeptide,and more preferably a yeast polypeptide such as a Candida,Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowiapolypeptide having enzyme activity; or more preferably a filamentousfungal polypeptide such as an Acremonium, Agaricus, Altemrnaria,Aspergillus, Aureobasidium, Botryosphaeria, Ceriporiopsis, Chaetomidium,Chrysosporium, Claviceps, Cochliobolus, Coprinopsis, Coptotermes,Corynascus, Cryphonectria, Cryptococcus, Diplodia, Exidia, Filibasidium,Fusarium, Gibberella, Holomastigotoides, Humicola, Irpex, Lentinula,Leptospaeria, Magnaporthe, Melanocarpus, Meripilus, Mucor,Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium,Phanerochaete, Piromyces, Poitrasia, Pseudoplectania,Pseudotrichonympha, Rhizomucor, Schizophyllum, Scytalidium, Talaromyces,Thermoascus, Thielavia, Tolypocladium, Trichoderma, Trichophaea,Verticillium, Volvatiella, or Xylaria polypeptide having enzymeactivity.

In one aspect, the polypeptide is a Saccharomyces carlsbergensis,Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomycesdouglasii, Saccharomyces kluyveri, Saccharomyces norbensis, orSaccharomyces oviformis polypeptide having enzyme activity.

In another aspect, the polypeptide is an Acremonium cellulolyticus,Aspergillus aculeatus, Aspergillus awamori, Aspergillus fumigatus,Aspergillus foetidus, Aspergillus japonicus, Aspergillus nidulans,Aspergillus niger, Aspergillus oryzae, Chrysosporium keratinophilum,Chrysosporium lucknowense, Chrysosporium tropicum, Chrysosporiummerdatium, Chrysosporium inops, Chrysosporium pannicola, Chrysosporiumqueenslandicum, Chrysosporium zonatum, Fusarium bactridioides, Fusariumcerealis, Fusarium crookwellense, Fusarium culmorum, Fusariumgraminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi,Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusariumsambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusariumsulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusariumvenenatum, Humicola grisea, Humicola insolens, Humicola lanuginosa,Irpex lacteus, Mucor miehei, Myceliophthora thermophila, Neurosporacrassa, Penicillium funiculosum, Penicillium purpurogenum, Phanerochaetechrysosporium, Thielavia achromatica, Thielavia albomyces, Thielaviaalbopilosa, Thielavia australeinsis, Thielavia fimeti, Thielaviamicrospora, Thielavia ovispora, Thielavia peruviana, Thielaviaspededonium, Thielavia setosa, Thielavia subthermophila, Thielaviaterrestris, Trichoderma harzianum, Trichoderma koningii, Trichodermalongibrachiatum, Trichoderma reesei, Trichoderma viride, or Trichophaeasaccata polypeptide having enzyme activity.

Chemically modified or protein engineered mutants of polypeptides havingenzyme activity may also be used.

One or more (e.g., several) components of the enzyme composition may bea recombinant component, i.e., produced by cloning of a DNA sequenceencoding the single component and subsequent cell transformed with theDNA sequence and expressed in a host (see, for example, WO 91/17243 andWO 91/17244). The host is preferably a heterologous host (enzyme isforeign to host), but the host may under certain conditions also be ahomologous host (enzyme is native to host). Monocomponent cellulolyticproteins may also be prepared by purifying such a protein from afermentation broth.

In one aspect, the one or more (e.g., several) cellulolytic enzymescomprise a commercial cellulolytic enzyme preparation. Examples ofcommercial cellulolytic enzyme preparations suitable for use in thepresent invention include, for example, CELLIC® CTec (Novozymes A/S),CELLIC® CTec2 (Novozymes A/S), CELLIC® CTec3 (Novozymes A/S),CELLUCLAST™ (Novozymes A/S), NOVOZYM™ 188 (Novozymes A/S), CELLUZYME™(Novozymes A/S), CEREFLO™ (Novozymes A/S), and ULTRAFLO™ (NovozymesA/S), ACCELERASE™ (Genencor Int.), LAMINEX™ (Genencor Int.), SPEZYME™ CP(Genencor Int.), FILTRASE® NL (DSM); METHAPLUS® S/L 100 (DSM). ROHAMENT™7069 W (Röhm GmbH), FIBREZYME® LDI (Dyadic International, Inc.),FIBREZYME® LBR (Dyadic International, Inc.), or VISCOSTAR® 150 L (DyadicInternational, Inc.). The cellulase enzymes are added in amountseffective from about 0.001 to about 5.0 wt % of solids, e.g., about0.025 to about 4.0 wt % of solids or about 0.005 to about 2.0 wt % ofsolids.

Examples of bacterial endoglucanases that can be used in the processesof the present invention, include, but are not limited to, anAcidothermus cellulolyticus endoglucanase (WO 91/05039; WO 93/15186;U.S. Pat. No. 5,275,944; WO 96/02551; U.S. Pat. No. 5,536,655, WO00/70031, WO 05/093050); Thermobifida fusca endoglucanase III (WO05/093050); and Thermobifida fusca endoglucanase V (WO 05/093050).

Examples of fungal endoglucanases that can be used in the presentinvention, include, but are not limited to, a Trichoderma reeseiendoglucanase I (Penttila et al., 1986, Gene 45: 253-263, Trichodermareesei CeI7B endoglucanase I (GENBANK™ accession no. M15665),Trichoderma reesei endoglucanase II (Saloheimo, et al., 1988, Gene63:11-22), Trichoderma reesei CeI5A endoglucanase II (GENBANK™ accessionno. M19373), Trichoderma reesei endoglucanase III (Okada et al., 1988,Appl. Environ. Microbiol. 64: 555-563, GENBANK™ accession no. AB003694),Trichoderma reesei endoglucanase V (Saloheimo et al., 1994, MolecularMicrobiology 13: 219-228, GENBANK™ accession no. Z33381), Aspergillusaculeatus endoglucanase (Ooi et al., 1990, Nucleic Acids Research 18:5884), Aspergillus kawachii endoglucanase (Sakamoto et al., 1995,Current Genetics 27: 435-439), Erwinia carotovara endoglucanase(Saarilahti et al., 1990, Gene 90: 9-14), Fusarium oxysporumendoglucanase (GENBANK™ accession no. L29381), Humicola grisea var.thermoidea endoglucanase (GENBANK™ accession no. AB003107), Melanocarpusalbomyces endoglucanase (GENBANK™ accession no. MAL515703), Neurosporacrassa endoglucanase (GENBANK™ accession no. XM_324477), Humicolainsolens endoglucanase V, Myceliophthora thermophila CBS 117.65endoglucanase, basidiomycete CBS 495.95 endoglucanase, basidiomycete CBS494.95 endoglucanase, Thielavia terrestris NRRL 8126 CEL6Bendoglucanase, Thielavia terrestris NRRL 8126 CEL6C endoglucanase,Thielavia terrestris NRRL 8126 CEL7C endoglucanase, Thielavia terrestrisNRRL 8126 CEL7E endoglucanase, Thielavia terrestris NRRL 8126 CEL7Fendoglucanase, Cladorrhinum foecundissimum ATCC 62373 CEL7Aendoglucanase, and Trichoderma reesei strain No. VTT-D-80133endoglucanase (GENBANK™ accession no. M15665).

Examples of cellobiohydrolases useful in the present invention include,but are not limited to, Aspergillus aculeatus cellobiohydrolase II (WO2011/059740), Chaetomium thermophilum cellobiohydrolase I, Chaetomiumthermophilum cellobiohydrolase II, Humicola insolens cellobiohydrolaseI, Myceliophthora thermophila cellobiohydrolase II (WO 2009/042871),Thielavia hyrcanie cellobiohydrolase II (WO 2010/141325), Thielaviaterrestris cellobiohydrolase II (CEL6A, WO 2006/074435), Trichodermareesei cellobiohydrolase I, Trichoderma reesei cellobiohydrolase II, andTrichophaea saccata cellobiohydrolase II (WO 2010/057086).

Examples of beta-glucosidases useful in the present invention include,but are not limited to, beta-glucosidases from Aspergillus aculeatus(Kawaguchi et al., 1996, Gene 173: 287-288), Aspergillus fumigatus (WO2005/047499), Aspergillus niger (Dan et al, 2000, J. Biol. Chem. 275:4973-4980), Aspergillus oryzae (WO 2002/095014), Penicillium brasilianumIBT 20888 (WO 2007/019442 and WO 2010/088387), Thielavia terrestris (WO2011/035029), and Trichophaea saccata (WO 2007/019442).

The beta-glucosidase may be a fusion protein. In one aspect, thebeta-glucosidase is an Aspergillus oryzae beta-glucosidase variant BGfusion protein (WO 2008/057637) or an Aspergillus oryzaebeta-glucosidase fusion protein (WO 2008/057637.

Other useful endoglucanases, cellobiohydrolases, and beta-glucosidasesare disclosed in numerous Glycosyl Hydrolase families using theclassification according to Henrissat B., 1991, A classification ofglycosyl hydrolases based on amino-acid sequence similarities, Biochem.J. 280: 309-316, and Henrissat B., and Bairoch A., 1996, Updating thesequence-based classification of glycosyl hydrolases, Biochem. J. 316:695-696.

Other cellulolytic enzymes that may be used in the present invention aredescribed in WO 98/13465, WO 98/015619, WO 98/015633, WO 99/06574, WO99/10481, WO 99/025847, WO 99/031255, WO 2002/101078, WO 2003/027306, WO2003/052054, WO 2003/052055, WO 2003/052056, WO 2003/052057, WO2003/052118, WO 2004/016760, WO 2004/043980, WO 2004/048592, WO2005/001065, WO 2005/028636, WO 2005/093050, WO 2005/093073, WO2006/074005, WO 2006/117432, WO 2007/071818, WO 2007/071820, WO2008/008070, WO 2008/008793, U.S. Pat. No. 5,457,046, U.S. Pat. No.5,648,263, and U.S. Pat. No. 5,686,593.

In the processes of the present invention, any GH61 polypeptide havingcellulolytic enhancing activity can be used.

Examples of GH61 polypeptides having cellulolytic enhancing activityuseful in the processes of the present invention include, but are notlimited to, GH61 polypeptides from Thielavia terrestris (WO 2005/074647,WO 2008/148131, and WO 2011/035027), Thermoascus aurantiacus (WO2005/074656 and WO 2010/065830), Trichoderma reesei (WO 2007/089290),Myceliophthora thermophila (WO 2009/085935, WO 2009/085859, WO2009/085864, WO 2009/085868), Aspergillus fumigatus (WO 2010/138754),GH61 polypeptides from Penicillium pinophilum (WO 2011/005867),Thermoascus sp. (WO 2011/039319), Penicillium sp. (WO 2011/041397), andThermoascus crustaceous (WO 2011/041504).

In one aspect, the GH61 polypeptide having cellulolytic enhancingactivity is used in the presence of a soluble activating divalent metalcation according to WO 2008/151043, e.g., manganese or copper.

In one aspect, the GH61 polypeptide having cellulolytic enhancingactivity is used in the presence of a dioxy compound, a bicyliccompound, a heterocyclic compound, a nitrogen-containing compound, aquinone compound, a sulfur-containing compound, or a liquor obtainedfrom a pretreated cellulosic material such as pretreated corn stover(PCS).

The dioxy compound may include any suitable compound containing two ormore oxygen atoms. In some aspects, the dioxy compounds contain asubstituted aryl moiety as described herein. The dioxy compounds maycomprise one or more (e.g., several) hydroxyl and/or hydroxylderivatives, but also include substituted aryl moieties lacking hydroxyland hydroxyl derivatives. Non-limiting examples of the dioxy compoundsinclude pyrocatechol or catechol; caffeic acid; 3,4-dihydroxybenzoicacid; 4-tert-butyl-5-methoxy-1,2-benzenediol; pyrogallol; gallic acid;methyl-3,4,5-trihydroxybenzoate; 2,3,4-trihydroxybenzophenone;2,6-dimethoxyphenol; sinapinic acid; 3,5-dihydroxybenzoic acid;4-chloro-1,2-benzenediol; 4-nitro-1,2-benzenediol; tannic acid; ethylgallate; methyl glycolate; dihydroxyfumaric acid; 2-butyne-1,4-diol;(croconic acid; 1,3-propanediol; tartaric acid; 2,4-pentanediol;3-ethyoxy-1,2-propanediol; 2,4,4′-trihydroxybenzophenone;cis-2-butene-1,4-diol; 3,4-dihydroxy-3-cyclobutene-1,2-dione;dihydroxyacetone; acrolein acetal; methyl-4-hydroxybenzoate;4-hydroxybenzoic acid; and methyl-3,5-dimethoxy-4-hydroxybenzoate; or asalt or solvate thereof.

The bicyclic compound may include any suitable substituted fused ringsystem as described herein. The compounds may comprise one or more(e.g., several) additional rings, and are not limited to a specificnumber of rings unless otherwise stated. In one aspect, the bicycliccompound is a flavonoid. In another aspect, the bicyclic compound is anoptionally substituted isoflavonoid. In another aspect, the bicycliccompound is an optionally substituted flavylium ion, such as anoptionally substituted anthocyanidin or optionally substitutedanthocyanin, or derivative thereof. Non-limiting examples of thebicyclic compounds include epicatechin; quercetin; myricetin; taxifolin;kaempferol; morin; acacetin; naringenin; isorhamnetin; apigenin;cyanidin; cyanin; kuromanin; keracyanin; or a salt or solvate thereof.

The heterocyclic compound may be any suitable compound, such as anoptionally substituted aromatic or non-aromatic ring comprising aheteroatom, as described herein. In one aspect, the heterocyclic is acompound comprising an optionally substituted heterocycloalkyl moiety oran optionally substituted heteroaryl moiety. In another aspect, theoptionally substituted heterocycloalkyl moiety or optionally substitutedheteroaryl moiety is an optionally substituted 5-memberedheterocycloalkyl or an optionally substituted 5-membered heteroarylmoiety. In another aspect, the optionally substituted heterocycloalkylor optionally substituted heteroaryl moiety is an optionally substitutedmoiety selected from pyrazolyl, furanyl, imidazolyl, isoxazolyl,oxadiazolyl, oxazolyl, pyrrolyl, pyridyl, pyrimidyl, pyridazinyl,thiazolyl, triazolyl, thienyl, dihydrothieno-pyrazolyl, thianaphthenyl,carbazolyl, benzimidazolyl, benzothienyl, benzofuranyl, indolyl,quinolinyl, benzotriazolyl, benzothiazolyl, benzooxazolyl,benzimidazolyl, isoquinolinyl, isoindolyl, acridinyl, benzoisazolyl,dimethylhydantoin, pyrazinyl, tetrahydrofuranyl, pyrrolinyl,pyrrolidinyl, morpholinyl, indolyl, diazepinyl, azepinyl, thiepinyl,piperidinyl, and oxepinyl. In another aspect, the optionally substitutedheterocycloalkyl moiety or optionally substituted heteroaryl moiety isan optionally substituted furanyl. Non-limiting examples of theheterocyclic compounds include(1,2-dihydroxyethyl)-3,4-dihydroxyfuran-2(5H)-one;4-hydroxy-5-methyl-3-furanone; 5-hydroxy-2(5H)-furanone;[1,2-dihydroxyethyl]furan-2,3,4(5H)-trione; α-hydroxy-γ-butyrolactone;ribonic γ-lactone; aldohexuronicaldohexuronic acid γ-lactone; gluconicacid δ-lactone; 4-hydroxycoumarin; dihydrobenzofuran;5-(hydroxymethyl)furfural; furoin; 2(5H)-furanone;5,6-dihydro-2H-pyran-2-one; and5,6-dihydro-4-hydroxy-6-methyl-2H-pyran-2-one; or a salt or solvatethereof.

The nitrogen-containing compound may be any suitable compound with oneor more nitrogen atoms. In one aspect, the nitrogen-containing compoundcomprises an amine, imine, hydroxylamine, or nitroxide moiety.Non-limiting examples of the nitrogen-containing compounds includeacetone oxime; violuric acid; pyridine-2-aldoxime; 2-aminophenol;1,2-benzenediamine; 2,2,6,6-tetramethyl-1-piperidinyloxy;5,6,7,8-tetrahydrobiopterin; 6,7-dimethyl-5,6,7,8-tetrahydropterine; andmaleamic acid; or a salt or solvate thereof.

The quinone compound may be any suitable compound comprising a quinonemoiety as described herein. Non-limiting examples of the quinonecompounds include 1,4-benzoquinone; 1,4-naphthoquinone;2-hydroxy-1,4-naphthoquinone; 2,3-dimethoxy-5-methyl-1,4-benzoquinone orcoenzyme Q₀; 2,3,5,6-tetramethyl-1,4-benzoquinone or duroquinone;1,4-dihydroxyanthraquinone; 3-hydroxy-1-methyl-5,6-indolinedione oradrenochrome; 4-tert-butyl-5-methoxy-1,2-benzoquinone; pyrroloquinolinequinone; or a salt or solvate thereof.

The sulfur-containing compound may be any suitable compound comprisingone or more sulfur atoms. In one aspect, the sulfur-containing comprisesa moiety selected from thionyl, thioether, sulfinyl, sulfonyl,sulfamide, sulfonamide, sulfonic acid, and sulfonic ester. Non-limitingexamples of the sulfur-containing compounds include ethanethiol;2-propanethiol; 2-propene-1-thiol; 2-mercaptoethanesulfonic acid;benzenethiol; benzene-1,2-dithiol; cysteine; methionine; glutathione;cystine; or a salt or solvate thereof.

In one aspect, an effective amount of such a compound described above tocellulosic material as a molar ratio to glucosyl units of cellulose isabout 10⁻⁶ to about 10, e.g., about 10⁻⁶ to about 7.5, about 10⁻⁶ toabout 5, about 10⁻⁶ to about 2.5, about 10⁻⁶ to about 1, about 10⁻⁵ toabout 1, about 10⁻⁵ to about 10⁻¹, about 10⁻⁴ to about 10⁻¹, about 10⁻³to about 10⁻¹, or about 10⁻³ to about 10⁻². In another aspect, aneffective amount of such a compound described above is about 0.1 μM toabout 1 M, e.g., about 0.5 μM to about 0.75 M, about 0.75 μM to about0.5 M, about 1 μM to about 0.25 M, about 1 μM to about 0.1 M, about 5 μMto about 50 mM, about 10 μM to about 25 mM, about 50 μM to about 25 mM,about 10 μM to about 10 mM, about 5 μM to about 5 mM, or about 0.1 mM toabout 1 mM.

The term “liquor” means the solution phase, either aqueous, organic, ora combination thereof, arising from treatment of a lignocellulose and/orhemicellulose material in a slurry, or monosaccharides thereof, e.g.,xylose, arabinose, mannose, etc., under conditions as described herein,and the soluble contents thereof. A liquor for cellulolytic enhancementof a GH61 polypeptide can be produced by treating a lignocellulose orhemicellulose material (or feedstock) by applying heat and/or pressure,optionally in the presence of a catalyst, e.g., acid, optionally in thepresence of an organic solvent, and optionally in combination withphysical disruption of the material, and then separating the solutionfrom the residual solids. Such conditions determine the degree ofcellulolytic enhancement obtainable through the combination of liquorand a GH61 polypeptide during hydrolysis of a cellulosic substrate by acellulase preparation. The liquor can be separated from the treatedmaterial using a method standard in the art, such as filtration,sedimentation, or centrifugation.

In one aspect, an effective amount of the liquor to cellulose is about10⁻⁶ to about 10 g per g of cellulose, e.g., about 10⁻⁶ to about 7.5 g,about 10⁻⁶ to about 5, about 10⁻⁶ to about 2.5 g, about 10⁻⁶ to about 1g, about 10⁻⁵ to about 1 g, about 10⁻⁵ to about 10⁻¹ g, about 10⁻⁴ toabout 10⁻¹ g, about 10⁻³ to about 10⁻¹ g, or about 10⁻³ to about 10⁻² gper g of cellulose.

In one aspect, the one or more (e.g., several) hemicellulolytic enzymescomprise a commercial hemicellulolytic enzyme preparation. Examples ofcommercial hemicellulolytic enzyme preparations suitable for use in thepresent invention include, for example, SHEARZYME™ (Novozymes A/S),CELLIC® HTec (Novozymes A/S), CELLIC® HTec2 (Novozymes A/S), CELLIC®HTec3 (Novozymes A/S), VISCOZYME® (Novozymes A/S), ULTRAFLO® (NovozymesA/S), PULPZYME® HC (Novozymes A/S), MULTIFECT® Xylanase (Genencor),ACCELLERASE® XY (Genencor), ACCELLERASE® XC (Genencor), ECOPULP® TX-200A(AB Enzymes), HSP 6000 Xylanase (DSM), DEPOL™ 333P (Biocatalysts Limit,Wales, UK), DEPOL™ 740 L (Biocatalysts Limit, Wales, UK), and DEPOL™762P (Biocatalysts Limit, Wales, UK).

Examples of xylanases useful in the processes of the present inventioninclude, but are not limited to, xylanases from Aspergillus aculeatus(GeneSeqP:AAR63790; WO 94/21785), Aspergillus fumigatus (WO2006/078256), Penicillium pinophilum (WO 2011/041405), Penicillium sp.(WO 2010/126772), Thielavia terrestris NRRL 8126 (WO 2009/079210), andTrichophaea saccata GH10 (WO 2011/057083).

Examples of beta-xylosidases useful in the processes of the presentinvention include, but are not limited to, beta-xylosidases fromNeurospora crassa (SwissProt accession number Q7SOW4), Trichodermareesei (UniProtKB/TrEMBL accession number Q92458), and Talaromycesemersonii (SwissProt accession number Q8X212).

Examples of acetylxylan esterases useful in the processes of the presentinvention include, but are not limited to, acetylxylan esterases fromAspergillus aculeatus (WO 2010/108918), Chaetomium globosum (Uniprotaccession number Q2GWX4), Chaetomium gracile (GeneSeqP accession numberAAB82124), Humicola insolens DSM 1800 (WO 2009/073709), Hypocreajecorina (WO 2005/001036), Myceliophtera thermophila (WO 2010/014880),Neurospora crassa (UniProt accession number q7s259), Phaeosphaerianodorum (Uniprot accession number QOUHJI), and Thielavia terrestris NRRL8126 (WO 2009/042846).

Examples of feruloyl esterases (ferulic acid esterases) useful in theprocesses of the present invention include, but are not limited to,feruloyl esterases form Humicola insolens DSM 1800 (WO 2009/076122),Neosartorya fischeri (UniProt Accession number A1D9T4), Neurosporacrassa (UniProt accession number Q9HGR3), Penicillium aurantiogriseum(WO 2009/127729), and Thielavia terrestris (WO 2010/053838 and WO2010/065448). Examples of arabinofuranosidases useful in the processesof the present invention include, but are not limited to,arabinofuranosidases from Aspergillus niger (GeneSeqP accession numberAAR94170), Humicola insolens DSM 1800 (WO 2006/114094 and WO2009/073383), and M. giganteus (WO 2006/114094).

Examples of alpha-glucuronidases useful in the processes of the presentinvention include, but are not limited to, alpha-glucuronidases fromAspergillus clavatus (UniProt accession number alcc12), Aspergillusfumigatus (SwissProt accession number Q4WW45), Aspergillus niger(Uniprot accession number Q96WX9), Aspergillus terreus (SwissProtaccession number QOCJP9), Humicola insolens (WO 2010/014706),Penicillium aurantiogriseum (WO 2009/068565), Talaromyces emersonii(UniProt accession number Q8X211), and Trichoderma reesei (Uniprotaccession number Q99024).

The polypeptides having enzyme activity used in the processes of thepresent invention may be produced by fermentation of the above-notedmicrobial strains on a nutrient medium containing suitable carbon andnitrogen sources and inorganic salts, using procedures known in the art(see, e.g., Bennett, J. W. and LaSure, L. (eds.), More GeneManipulations in Fungi, Academic Press, CA, 1991). Suitable media areavailable from commercial suppliers or may be prepared according topublished compositions (e.g., in catalogues of the American Type CultureCollection). Temperature ranges and other conditions suitable for growthand enzyme production are known in the art (see, e.g., Bailey, J. E.,and Ollis, D. F., Biochemical Engineering Fundamentals, McGraw-Hill BookCompany, NY, 1986).

The fermentation can be any method of cultivation of a cell resulting inthe expression or isolation of an enzyme or protein. Fermentation may,therefore, be understood as comprising shake flask cultivation, orsmall- or large-scale fermentation (including continuous, batch,fed-batch, or solid state fermentations) in laboratory or industrialfermentors performed in a suitable medium and under conditions allowingthe enzyme to be expressed or isolated. The resulting enzymes producedby the methods described above may be recovered from the fermentationmedium and purified by conventional procedures.

Fermentation. The fermentable sugars obtained from the hydrolyzedcellulosic material can be fermented by one or more (e.g., several)fermenting microorganisms capable of fermenting the sugars directly orindirectly into a desired fermentation product. “Fermentation” or“fermentation process” refers to any fermentation process or any processcomprising a fermentation step. Fermentation processes also includefermentation processes used in the consumable alcohol industry (e.g.,beer and wine), dairy industry (e.g., fermented dairy products), leatherindustry, and tobacco industry. The fermentation conditions depend onthe desired fermentation product and fermenting organism and can easilybe determined by one skilled in the art.

In the fermentation step, sugars, released from the cellulosic materialas a result of the pretreatment and enzymatic hydrolysis steps, arefermented to a product, e.g., ethanol, by a fermenting organism, such asyeast. Hydrolysis (saccharification) and fermentation can be separate orsimultaneous, as described herein.

Any suitable hydrolyzed cellulosic material can be used in thefermentation step in practicing the present invention. The material isgenerally selected based on the desired fermentation product, i.e., thesubstance to be obtained from the fermentation, and the processemployed, as is well known in the art.

The term “fermentation medium” is understood herein to refer to a mediumbefore the fermenting microorganism(s) is (are) added, such as, a mediumresulting from a saccharification process, as well as a medium used in asimultaneous saccharification and fermentation process (SSF).

“Fermenting microorganism” refers to any microorganism, includingbacterial and fungal organisms, suitable for use in a desiredfermentation process to produce a fermentation product. The fermentingorganism can be hexose and/or pentose fermenting organisms, or acombination thereof. Both hexose and pentose fermenting organisms arewell known in the art. Suitable fermenting microorganisms are able toferment, i.e., convert, sugars, such as glucose, xylose, xylulose,arabinose, maltose, mannose, galactose, and/or oligosaccharides,directly or indirectly into the desired fermentation product. Examplesof bacterial and fungal fermenting organisms producing ethanol aredescribed by Lin et al, 2006, Appl. Microbiol. Biotechnol. 69: 627-642.

Examples of fermenting microorganisms that can ferment hexose sugarsinclude bacterial and fungal organisms, such as yeast. Preferred yeastincludes strains of Candida, Kluyveromyces, and Saccharomyces, e.g.,Candida sonorensis, Kluyveromyces marxianus, and Saccharomycescerevisiae.

Examples of fermenting organisms that can ferment pentose sugars intheir native state include bacterial and fungal organisms, such as someyeast. Preferred xylose fermenting yeast include strains of Candida,preferably C. sheatae or C. sonorensis; and strains of Pichia,preferably P. stipitis, such as P. stipitis CBS 5773. Preferred pentosefermenting yeast include strains of Pachysolen, preferably P.tannophilus. Organisms not capable of fermenting pentose sugars, such asxylose and arabinose, may be genetically modified to do so by methodsknown in the art

Examples of bacteria that can efficiently ferment hexose and pentose toethanol include, for example, Bacillus coagulans, Clostridiumacetobutylicum, Clostridium thermocellum, Clostridium phytofermentans,Geobacillus sp., Thermoanaerobacter saccharolyticum, and Zymomonasmobilis (Philippidis, 1996, supra).

Other fermenting organisms include strains of Bacillus, such as Bacilluscoagulans; Candida, such as C. sonorensis, C. methanosorbosa, C.diddensiae, C. parapsilosis, C. naedodendra, C. blankii, C.entomophilia, C. brassicae, C. pseudotropicalis, C. boidinii, C. utilis,and C. scehatae; Clostridium, such as C. acetobutylicum, C.thermocellum, and C. phytofermentans; E. coli, especially E. colistrains that have been genetically modified to improve the yield ofethanol; Geobacillus sp.; Hansenula, such as Hansenula anomala;Klebsiella, such as K. oxytoca; Kluyveromyces, such as K. marxianus, K.lactis, K. thermotolerans, and K. fragilis; Schizosaccharomyces, such asS. pombe; Thermoanaerobacter, such as Thermoanaerobactersaccharolyticum; and Zymomonas, such as Zymomonas mobilis.

In a preferred aspect, the yeast is a Bretannomyces. In a more preferredaspect, the yeast is Bretannomyces clausenii. In another preferredaspect, the yeast is a Candida. In another more preferred aspect, theyeast is Candida sonorensis. In another more preferred aspect, the yeastis Candida boidinii. In another more preferred aspect, the yeast isCandida blankii. In another more preferred aspect, the yeast is Candidabrassicae. In another more preferred aspect, the yeast is Candidadiddensii. In another more preferred aspect, the yeast is Candidaentomophiliia. In another more preferred aspect, the yeast is Candidapseudotropicalis. In another more preferred aspect, the yeast is Candidascehatae. In another more preferred aspect, the yeast is Candida utilis.In another preferred aspect, the yeast is a Clavispora. In another morepreferred aspect, the yeast is Clavispora lusitaniae. In another morepreferred aspect, the yeast is Clavispora opuntiae. In another preferredaspect, the yeast is a Kluyveromyces. In another more preferred aspect,the yeast is Kluyveromyces fragilis. In another more preferred aspect,the yeast is Kluyveromyces marxianus. In another more preferred aspect,the yeast is Kluyveromyces thermotolerans. In another preferred aspect,the yeast is a Pachysolen. In another more preferred aspect, the yeastis Pachysolen tannophilus. In another preferred aspect, the yeast is aPichia. In another more preferred aspect, the yeast is a Pichiastipitis. In another preferred aspect, the yeast is a Saccharomyces spp.In a more preferred aspect, the yeast is Saccharomyces cerevisiae. Inanother more preferred aspect, the yeast is Saccharomyces distaticus. Inanother more preferred aspect, the yeast is Saccharomyces uvarum.

In a preferred aspect, the bacterium is a Bacillus. In a more preferredaspect, the bacterium is Bacillus coagulans. In another preferredaspect, the bacterium is a Clostridium. In another more preferredaspect, the bacterium is Clostridium acetobutylicum. In another morepreferred aspect, the bacterium is Clostridium phytofermentans. Inanother more preferred aspect, the bacterium is Clostridiumthermocellum. In another more preferred aspect, the bacterium isGeobacillus sp. In another more preferred aspect, the bacterium is aThermoanaerobacter. In another more preferred aspect, the bacterium isThermoanaerobacter saccharolyticum. In another preferred aspect, thebacterium is a Zymomonas. In another more preferred aspect, thebacterium is Zymomonas mobilis.

Commercially available yeast suitable for ethanol production include,e.g., BIOFERM™ AFT and XR (NABC—North American Bioproducts Corporation,GA, USA), ETHANOL RED™ yeast (Fermentis/Lesaffre, USA), FALI™(Fleischmann's Yeast, USA), FERMIOL™ (DSM Specialties), GERT STRAND™(Gert Strand AB, Sweden), and SUPERSTART™ and THERMOSACC™ fresh yeast(Ethanol Technology, WI, USA).

In a preferred aspect, the fermenting microorganism has been geneticallymodified to provide the ability to ferment pentose sugars, such asxylose utilizing, arabinose utilizing, and xylose and arabinoseco-utilizing microorganisms.

The cloning of heterologous genes into various fermenting microorganismshas led to the construction of organisms capable of converting hexosesand pentoses to ethanol (co-fermentation) (Chen and Ho, 1993, Cloningand improving the expression of Pichia stipitis xylose reductase gene inSaccharomyces cerevisiae, Appl. Biochem. Biotechnol 39-40: 135-147; Hoet al., 1998, Genetically engineered Saccharomyces yeast capable ofeffectively cofermenting glucose and xylose, Appl. Environ. Microbiol.64: 1852-1859; Kotter and Ciriacy, 1993, Xylose fermentation bySaccharomyces cerevisiae, Appl. Microbiol. Biotechnol 38: 776-783;Walfridsson et al., 1995, Xylose-metabolizing Saccharomyces cerevisiaestrains overexpressing the TKL1 and TAL1 genes encoding the pentosephosphate pathway enzymes transketolase and transaldolase, Appl.Environ. Microbiol. 61: 4184-4190; Kuyper et al., 2004, Minimalmetabolic engineering of Saccharomyces cerevisiae for efficientanaerobic xylose fermentation: a proof of principle, FEMS Yeast Research4: 655-664; Beall et al, 1991, Parametric studies of ethanol productionfrom xylose and other sugars by recombinant Escherichia coli, Biotech.Bioeng. 38: 296-303; Ingram et al., 1998, Metabolic engineering ofbacteria for ethanol production, Biotechnol. Bioeng. 58: 204-214; Zhanget al., 1995, Metabolic engineering of a pentose metabolism pathway inethanologenic Zymomonas mobilis, Science 267: 240-243; Deanda et al.,1996, Development of an arabinose-fermenting Zymomonas mobilis strain bymetabolic pathway engineering, Appl. Environ. Microbiol. 62: 4465-4470;WO 2003/062430, xylose isomerase).

In a preferred aspect, the genetically modified fermenting microorganismis Candida sonorensis. In another preferred aspect, the geneticallymodified fermenting microorganism is Escherichia coli. In anotherpreferred aspect, the genetically modified fermenting microorganism isKlebsiella oxytoca. In another preferred aspect, the geneticallymodified fermenting microorganism is Kluyveromyces marxianus. In anotherpreferred aspect, the genetically modified fermenting microorganism isSaccharomyces cerevisiae. In another preferred aspect, the geneticallymodified fermenting microorganism is Zymomonas mobilis.

It is well known in the art that the organisms described above can alsobe used to produce other substances, as described herein.

The fermenting microorganism is typically added to the degradedcellulosic material or hydrolysate and the fermentation is performed forabout 8 to about 96 hours, e.g., about 24 to about 60 hours. Thetemperature is typically between about 26° C. to about 60° C., e.g.,about 32° C. or 50° C., and about pH 3 to about pH 8, e.g., pH 4-5, 6,or 7.

In one aspect, the yeast and/or another microorganism are applied to thedegraded cellulosic material and the fermentation is performed for about12 to about 96 hours, such as typically 24-60 hours. In another aspect,the temperature is preferably between about 20° C. to about 60° C.,e.g., about 25° C. to about 50° C., about 32° C. to about 50° C., orabout 32° C. to about 50° C., and the pH is generally from about pH 3 toabout pH 7, e.g., about pH 4 to about pH 7. However, some fermentingorganisms, e.g., bacteria, have higher fermentation temperature optima.Yeast or another microorganism is preferably applied in amounts ofapproximately 10⁵ to 10¹², preferably from approximately 10⁷ to 10¹⁰,especially approximately 2×10⁸ viable cell count per ml of fermentationbroth. Further guidance in respect of using yeast for fermentation canbe found in, e.g., “The Alcohol Textbook” (Editors K. Jacques, T. P.Lyons and D. R. Kelsall, Nottingham University Press, United Kingdom1999), which is hereby incorporated by reference.

A fermentation stimulator can be used in combination with any of theprocesses described herein to further improve the fermentation process,and in particular, the performance of the fermenting microorganism, suchas, rate enhancement and ethanol yield. A “fermentation stimulator”refers to stimulators for growth of the fermenting microorganisms, inparticular, yeast. Preferred fermentation stimulators for growth includevitamins and minerals. Examples of vitamins include multivitamins,biotin, pantothenate, nicotinic acid, meso-inositol, thiamine,pyridoxine, para-aminobenzoic acid, folic acid, riboflavin, and VitaminsA, B, C, D, and E. See, for example, Alfenore et al, Improving ethanolproduction and viability of Saccharomyces cerevisiae by a vitaminfeeding strategy during fed-batch process, Springer-Verlag (2002), whichis hereby incorporated by reference. Examples of minerals includeminerals and mineral salts that can supply nutrients comprising P, K,Mg, S, Ca, Fe, Zn, Mn, and Cu.

Fermentation Products: A fermentation product can be any substancederived from the fermentation. The fermentation product can be, withoutlimitation, an alcohol (e.g., arabinitol, n-butanol, isobutanol,ethanol, glycerol, methanol, ethylene glycol, 1,3-propanediol [propyleneglycol], butanediol, glycerin, sorbitol, and xylitol); an alkane (e.g.,pentane, hexane, heptane, octane, nonane, decane, undecane, anddodecane), a cycloalkane (e.g., cyclopentane, cyclohexane, cycloheptane,and cyclooctane), an alkene (e.g. pentene, hexene, heptene, and octene);an amino acid (e.g., aspartic acid, glutamic acid, glycine, lysine,serine, and threonine); a gas (e.g., methane, hydrogen (H₂), carbondioxide (CO₂), and carbon monoxide (CO)); isoprene; a ketone (e.g.,acetone); an organic acid (e.g., acetic acid, acetonic acid, adipicacid, ascorbic acid, citric acid, 2,5-diketo-D-gluconic acid, formicacid, fumaric acid, glucaric acid, gluconic acid, glucuronic acid,glutaric acid, 3-hydroxypropionic acid, itaconic acid, lactic acid,malic acid, malonic acid, oxalic acid, oxaloacetic acid, propionic acid,succinic acid, and xylonic acid); and polyketide. The fermentationproduct can also be protein as a high value product.

In a preferred aspect, the fermentation product is an alcohol. It willbe understood that the term “alcohol” encompasses a substance thatcontains one or more hydroxyl moieties. In a more preferred aspect, thealcohol is n-butanol. In another more preferred aspect, the alcohol isisobutanol. In another more preferred aspect, the alcohol is ethanol. Inanother more preferred aspect, the alcohol is methanol. In another morepreferred aspect, the alcohol is arabinitol. In another more preferredaspect, the alcohol is butanediol. In another more preferred aspect, thealcohol is ethylene glycol. In another more preferred aspect, thealcohol is glycerin. In another more preferred aspect, the alcohol isglycerol. In another more preferred aspect, the alcohol is1,3-propanediol. In another more preferred aspect, the alcohol issorbitol. In another more preferred aspect, the alcohol is xylitol. See,for example, Gong, C. S., Cao, N. J., Du, J., and Tsao, G. T., 1999,Ethanol production from renewable resources, in Advances in BiochemicalEngineering/Biotechnology, Scheper, T., ed., Springer-Verlag BerlinHeidelberg, Germany, 65: 207-241; Silveira, M. M., and Jonas, R., 2002,The biotechnological production of sorbitol, Appl. Microbiol.Biotechnol. 59: 400-408; Nigam, P., and Singh, D., 1995, Processes forfermentative production of xylitol—a sugar substitute, ProcessBiochemistry 30 (2): 117-124; Ezeji, T. C., Qureshi, N. and Blaschek, H.P., 2003, Production of acetone, butanol and ethanol by Clostridiumbeijerinckii BA101 and in situ recovery by gas stripping, World Journalof Microbiology and Biotechnology 19 (6): 595-603.

In another preferred aspect, the fermentation product is an alkane. Thealkane can be an unbranched or a branched alkane. In another morepreferred aspect, the alkane is pentane. In another more preferredaspect, the alkane is hexane. In another more preferred aspect, thealkane is heptane. In another more preferred aspect, the alkane isoctane. In another more preferred aspect, the alkane is nonane. Inanother more preferred aspect, the alkane is decane. In another morepreferred aspect, the alkane is undecane. In another more preferredaspect, the alkane is dodecane.

In another preferred aspect, the fermentation product is a cycloalkane.In another more preferred aspect, the cycloalkane is cyclopentane. Inanother more preferred aspect, the cycloalkane is cyclohexane. Inanother more preferred aspect, the cycloalkane is cycloheptane. Inanother more preferred aspect, the cycloalkane is cyclooctane.

In another preferred aspect, the fermentation product is an alkene. Thealkene can be an unbranched or a branched alkene. In another morepreferred aspect, the alkene is pentene. In another more preferredaspect, the alkene is hexene. In another more preferred aspect, thealkene is heptene. In another more preferred aspect, the alkene isoctene.

In another preferred aspect, the fermentation product is an amino acid.In another more preferred aspect, the organic acid is aspartic acid. Inanother more preferred aspect, the amino acid is glutamic acid. Inanother more preferred aspect, the amino acid is glycine. In anothermore preferred aspect, the amino acid is lysine. In another morepreferred aspect, the amino acid is serine. In another more preferredaspect, the amino acid is threonine. See, for example, Richard, A., andMargaritis, A., 2004, Empirical modeling of batch fermentation kineticsfor poly(glutamic acid) production and other microbial biopolymers,Biotechnology and Bioengineering 87 (4): 501-515.

In another preferred aspect, the fermentation product is a gas. Inanother more preferred aspect, the gas is methane. In another morepreferred aspect, the gas is H₂. In another more preferred aspect, thegas is CO₂. In another more preferred aspect, the gas is CO. See, forexample, Kataoka, N., A. Miya, and K. Kiriyama, 1997, Studies onhydrogen production by continuous culture system of hydrogen-producinganaerobic bacteria, Water Science and Technology 36 (6-7): 41-47; andGunaseelan V. N. in Biomass and Bioenergy, Vol. 13 (1-2), pp. 83-114,1997, Anaerobic digestion of biomass for methane production: A review.

In another preferred aspect, the fermentation product is isoprene.

In another preferred aspect, the fermentation product is a ketone. Itwill be understood that the term “ketone” encompasses a substance thatcontains one or more ketone moieties. In another more preferred aspect,the ketone is acetone. See, for example, Qureshi and Blaschek, 2003,supra.

In another preferred aspect, the fermentation product is an organicacid. In another more preferred aspect, the organic acid is acetic acid.In another more preferred aspect, the organic acid is acetonic acid. Inanother more preferred aspect, the organic acid is adipic acid. Inanother more preferred aspect, the organic acid is ascorbic acid. Inanother more preferred aspect, the organic acid is citric acid. Inanother more preferred aspect, the organic acid is 2,5-diketo-D-gluconicacid. In another more preferred aspect, the organic acid is formic acid.In another more preferred aspect, the organic acid is fumaric acid. Inanother more preferred aspect, the organic acid is glucaric acid. Inanother more preferred aspect, the organic acid is gluconic acid. Inanother more preferred aspect, the organic acid is glucuronic acid. Inanother more preferred aspect, the organic acid is glutaric acid. Inanother preferred aspect, the organic acid is 3-hydroxypropionic acid.In another more preferred aspect, the organic acid is itaconic acid. Inanother more preferred aspect, the organic acid is lactic acid. Inanother more preferred aspect, the organic acid is malic acid. Inanother more preferred aspect, the organic acid is malonic acid. Inanother more preferred aspect, the organic acid is oxalic acid. Inanother more preferred aspect, the organic acid is propionic acid. Inanother more preferred aspect, the organic acid is succinic acid. Inanother more preferred aspect, the organic acid is xylonic acid. See,for example, Chen, R., and Lee, Y. Y., 1997, Membrane-mediatedextractive fermentation for lactic acid production from cellulosicbiomass, Appl. Biochem. Biotechnol. 63-65: 435-448.

In another preferred aspect, the fermentation product is polyketide.

Recovery. The fermentation product(s) can be optionally recovered fromthe fermentation medium using any method known in the art including, butnot limited to, chromatography, electrophoretic procedures, differentialsolubility, distillation, or extraction. For example, alcohol isseparated from the fermented cellulosic material and purified byconventional methods of distillation. Ethanol with a purity of up toabout 96 vol. % can be obtained, which can be used as, for example, fuelethanol, drinking ethanol, i.e., potable neutral spirits, or industrialethanol.

Signal Peptide

The present invention also relates to isolated polynucleotides encodinga signal peptide comprising or consisting of amino acids 1 to 19 of SEQID NO: 8, amino acids 1 to 16 of SEQ ID NO: 2, amino acids 1 to 20 ofSEQ ID NO: 4, or amino acids 1 to 24 of SEQ ID NO: 6. The polynucleotidemay further comprise a gene encoding a protein, which is operably linkedto the signal peptide. The protein is preferably foreign to the signalpeptide. In another aspect, the polynucleotide encoding the signalpeptide is nucleotides 1 to 57 of SEQ ID NO: 7. In one aspect, thepolynucleotide encoding the signal peptide is nucleotides 1 to 48 of SEQID NO: 1. In another aspect, the polynucleotide encoding the signalpeptide is nucleotides 1 to 60 of SEQ ID NO: 3. In another aspect, thepolynucleotide encoding the signal peptide is nucleotides 1 to 72 of SEQID NO: 5.

The present invention also relates to nucleic acid constructs,expression vectors and recombinant host cells comprising suchpolynucleotides.

The present invention also relates to methods of producing a protein,comprising (a) cultivating a recombinant host cell comprising such apolynucleotide operably linked to a gene encoding the protein; andoptionally (b) recovering the protein.

The protein may be native or heterologous to a host cell. The term“protein” is not meant herein to refer to a specific length of theencoded product and, therefore, encompasses peptides, oligopeptides, andpolypeptides. The term “protein” also encompasses two or morepolypeptides combined to form the encoded product. The proteins alsoinclude hybrid polypeptides and fused polypeptides.

Preferably, the protein is a hormone, enzyme, receptor or portionthereof, antibody or portion thereof, or reporter. For example, theprotein may be a hydrolase, isomerase, ligase, lyase, oxidoreductase, ortransferase, e.g., an alpha-galactosidase, alpha-glucosidase,aminopeptidase, amylase, beta-galactosidase, beta-glucosidase,beta-xylosidase, carbohydrase, carboxypeptidase, catalase,cellobiohydrolase, cellulase, chitinase, cutinase, cyclodextringlycosyltransferase, deoxyribonuclease, endoglucanase, esterase,glucoamylase, invertase, laccase, lipase, mannosidase, mutanase,oxidase, pectinolytic enzyme, peroxidase, phytase, polyphenoloxidase,proteolytic enzyme, ribonuclease, transglutaminase, or xylanase.

The gene may be obtained from any prokaryotic, eukaryotic, or othersource.

The present invention is further described by the following examplesthat should not be construed as limiting the scope of the invention.

EXAMPLES

Strain

The fungal strain NN044758 was isolated from a soil sample collectedfrom Yunnan Province in China by the dilution plate method with PDAmedium at 45° C. It was then purified by transferring a single conidiumonto a YG agar plate. The strain NN044758 was identified as Malbrancheacinnamomea, based on both morphological characteristics and ITS rDNAsequence.

The fungal strain NN046782 was isolated from a soil sample collectedfrom Hunan Province in China. The strain NN046872 was identified asRhizomucor pusillus, based on both morphological characteristics and ITSrDNA sequence.

The fungal strain NN051602 was isolated from a compost sample collectedfrom Yunnan Province, China by the dilution plate method with PDA mediumat 45° C. It was then purified by transferring a single conidium onto aYG agar plate. The strain NN051602 was identified as Penicilliumemersonii, based on both morphological characteristics and ITS rDNAsequence.

Media

PDA medium was composed of 39 grams of potato dextrose agar anddeionized water to 1 liter.

YG agar plate was composed of 5.0 g of yeast extract, 10.0 g of glucose,20.0 g of agar, and deionized water to 1 liter.

YPG medium was composed of 0.4% of yeast extract, 0.1% of KH₂PO₄, 0.05%of MgSO₄.7H₂O, 1.5% glucose in deionized water.

YPM medium was composed of 1% yeast extract, 2% of peptone, and 2% ofmaltose in deionized water.

Minimal medium plates were composed of 342 g of sucrose, 20 ml of saltsolution, 20 g of agar, and deionized water to 1 liter. The saltsolution was composed of 2.6% KCl, 2.6% MgSO₄.7H₂O, 7.6% KH₂PO₄, 2 ppmNa₂B₄O₇.10H₂O, 20 ppm CuSO₄.5H₂O, 40 ppm FeSO₄.7H₂O, 40 ppm MnSO₄.2H₂O,40 ppm Na₂MoO₄.2H₂O, and 400 ppm ZnSO₄.7H₂O.

FG4 medium was composed of 30 g of soymeal, 15 g of maltose, 5 g ofpeptone, and deionized water to 1 liter.

Example 1 Malbranchea cinnamomea Genomic DNA Extraction

Malbranchea cinnamomea strain NN044758 was inoculated onto a PDA plateand incubated for 3 days at 45° C. in the darkness. Several mycelia-PDAplugs were inoculated into 500 ml shake flasks containing 100 ml of YPGmedium. The flasks were incubated for 3 days at 45° C. with shaking at160 rpm. The mycelia were collected by filtration through MIRACLOTH®(Calbiochem, La Jolla, Calif., USA) and frozen under liquid nitrogen.Frozen mycelia were ground, by a mortar and a pestle, to a fine powder,and genomic DNA was isolated using Large-Scale Column Fungal DNAout(BAOMAN BIOTECHNOLOGY, Shanghai, China) following the manufacturer'sinstruction.

Example 2 Genome Sequencing, Assembly and Annotation

The extracted genomic DNA samples were delivered to Beijing GenomeInstitute (BGI, Shenzhen, China) for genome sequencing using anILLUMINA® GA2 System (Illumina, Inc., San Diego, Calif., USA). The rawreads were assembled at BGI using program SOAPdenovo (Li et al., 2010,Genome Research 20(2): 265-72). The assembled sequences were analyzedusing standard bioinformatics methods for gene identification andfunctional prediction. GenelD (Parra et al., 2000, Genome Research10(4):511-515) was used for gene prediction. Blastall version 2.2.10(Altschul et al., 1990, J. Mol. Biol. 215 (3): 403-410, National Centerfor Biotechnology Information (NCBI), Bethesda, Md., USA) and HMMERversion 2.1.1 (National Center for Biotechnology Information (NCBI),Bethesda, Md., USA) were used to predict function based on structuralhomology. The catalase was identified by analysis of the Blast results.The Agene program (Munch and Krogh, 2006, BMC Bioinformatics 7: 263) andSignalP program (Nielsen et al., 1997, Protein Engineering 10: 1-6) wereused to identify starting codons. The SignalP program was further usedto predict signal peptide. Pepstats (Rice et al, 2000, Trends Genet.16(6): 276-277) was used to predict isoelectric point and molecularweight of the deduced amino acid sequence.

Example 3 Cloning of the Malbranchea cinnamomea Catalase Gene fromGenomic DNA

One catalase gene, cat_ZY582303_121 (SEQ ID NO: 1), was selected forexpression cloning.

Based on DNA information (SEQ ID NO: 1) obtained from genome sequencing,oligonucleotide primers, shown below in Table 1, were designed toamplify the catalase gene from the genomic DNA of Malbranchea cinnamomeaNN044758. Primers were synthesized by Invitrogen Beijing, China.

TABLE 1 primers Forward primer ACACAACTGGGGATCC ACC SEQ ID NO: 9atgccgaacctcgtacgg Reverse primer GTCACCCTCTAGATCT gaag SEQ ID NO: 10gtgcactactgaccttacac  gag

Lowercase characters of the forward primer represent the coding regionsof the gene and lowercase characters of the reverse primer represent theflanking region of the gene, while capitalized parts were homologous tothe insertion sites of pPFJO355 vector which has been described inWO2011005867.

Twenty picomoles of each forward and reverse primer pair were used in aPCR reaction composed of 2 μl of Malbranchea cinnamomea NN044758 genomicDNA, 10 μl of 5×GC Buffer, 1.5 μl of dimethyl sulphoxide (DMSO), 2.5 mMeach of dATP, dTTP, dGTP, and dCTP, and 0.6 unit of PHUSION™High-Fidelity DNA Polymerase (Finnzymes Oy, Espoo, Finland) in a finalvolume of 50 μl. The amplification was performed using a Peltier ThermalCycler (M J Research Inc., South San Francisco, Calif., USA) programmedfor denaturing at 94° C. for 1 minute; 6 cycles of denaturing at 94° C.for 15 seconds, annealing at 68° C. for 30 seconds, with a 1° C.decrease per cycle and elongation at 72° C. for 100 seconds; and another23 cycles each at 94° C. for 15 seconds, 65° C. for 30 seconds and 72°C. for 100 seconds; and a final extension at 72° C. for 5 minutes. Theheat block then went to a 4° C. soak cycle.

The PCR product was isolated by 1.0% agarose gel electrophoresis using90 mM Tris-borate and 1 mM EDTA (TBE) buffer where a single product bandaround the expected size, ˜2.6 kb, was visualized under UV light. PCRproduct was then purified from solution by using an ILLUSTRA® GFX® PCRDNA and Gel Band Purification Kit (GE Healthcare, Buckinghamshire, UK)according to the manufacturer's instructions.

Plasmid pPFJO355 was digested with Bam HI and Bgl II, isolated by 1.0%agarose gel electrophoresis using TBE buffer, and purified using anILLUSTRA® GFX® PCR DNA and Gel Band Purification Kit according to themanufacturer's instructions.

An IN-FUSION™ CF Dry-down Cloning Kit (Clontech Laboratories, Inc.,Mountain View, Calif., USA) was used to clone the fragment directly intothe expression vector pPFJO355, without the need for restrictiondigestion and ligation.

The PCR product and the digested vector were ligated together using anIN-FUSION™ CF Dry-down PCR Cloning Kit (Clontech Laboratories, Inc.,Mountain View, Calif., USA) resulting in plasmid pCat_ZY582303_121 (FIG.5) in which the transcription of Malbranchea cinnamomea catalase genewas under the control of a promoter from the gene for Aspergillus oryzaealpha-amylase. The cloning operation was conducted according to themanufacturer's instruction. In brief, for each ligation reaction 30 ngof pPFJO355 digested with Bam HI and Bgl II, and 60 ng of the purifiedMalbranchea cinnamomea catalase PCR products were added to the reactionvials and resuspended the powder in a final volume of 10 μl withaddition of deionized water. The reaction was incubated at 37° C. for 15minutes and then 50° C. for 15 minutes. Three microlitres of thereaction products were used to transform E. coli TOP10 competent cells(TIANGEN Biotech (Beijing) Co. Ltd., Beijing, China). E. colitransformants containing expression constructs were detected by colonyPCR which is a method for quick screening of plasmid inserts directlyfrom E. coli colonies. Briefly, in the premixed PCR solution aliquot ineach PCR tube, including PCR buffer, MgCl₂, dNTP and primer pairs forwhich the PCR fragment generated, a single colony was added by pickingup with a sterile tip and twirling the tip in the reaction solution.Normaly 7-10 colonies were screened. After the PCR program, reactionswere checked on agarose gel. The colony giving the amplification ofexpected size was possibly to contain the correct insert. Plasmid DNAwas prepared from colonies showing inserts with the expected sizes usinga QIAprep® Spin Miniprep Kit (QIAGEN GmbH, Hilden, Germany). TheMalbranchea cinnamomea catalase gene inserted in pCat_ZY582303_121 wasconfirmed by DNA sequencing using a 3730XL DNA Analyzer (AppliedBiosystems Inc, Foster City, Calif., USA).

Example 4 Expression of Malbranchea cinnamomea catalase gene inAspergillus oryzae

Aspergillus oryzae HowB101 (described in patent WO9535385 example 1)protoplasts were prepared according to the method of Christensen et al,1988, Bio/Technology 6: 1419-1422 and transformed with 3 μg ofpCat_ZY582303_121.

The transformation of Aspergillus oryzae HowB101 with pCat_ZY582303_121yielded about 50 transformants for each transformation. Eighttransformants were isolated to individual Minimal medium plates.

Four transformants from the transformation were inoculated separatelyinto 3 ml of YPM medium in a 24-well plate and incubated at 30° C. withmixing at 150 rpm. After 3 days incubation, 20 μl of supernatant fromeach culture were analyzed by SDS-PAGE using a NUPAGE® NOVEX® 4-12%Bis-Tris Gel with 50 mM MES (Invitrogen Corporation, Carlsbad, Calif.,USA) according to the manufacturer's instructions. The resulting gel wasstained with INSTANTBLUE™ (Expedeon Ltd., Babraham Cambridge, UK).SDS-PAGE profiles of the cultures showed the expression with proteinbands detected. The size of major band of the gene was around 80 kDa.The expression strain was designated as O6QZB.

Example 5 Fermentation of Expression Strain O6QZB

A slant of the expression strain, O6QZB, was washed with 10 ml of YPMmedium and inoculated into eight 2-liter flasks, each containing 400 mlof YPM medium to generate broth. The culture was harvested on day 3 andfiltered using a 0.45 μm DURAPORE® Membrane (Millipore, Bedford, Mass.,USA).

Example 6 Purification of Recombinant Ma/Branchea Cinnamomea Catalasefrom Aspergillus orzyae O6QZB

3200 ml volume of filtered supernatant of the recombinant strain O6QZB(Example 5) was precipitated with ammonium sulfate (80% saturation),re-dissolved in 50 ml 20 mM Bis-Tris buffer, pH6.5, dialyzed against thesame buffer, and filtered through a 0.45 μm filter. The final volume was80 ml. The solution was applied to a 40 ml Q SEPHAROSE® Fast Flow column(GE Healthcare, Buckinghamshire, UK) equilibrated in 20 mM Bis-Trisbuffer, pH6.5, and the proteins was eluted with a linear NaCl gradient(0-0.5M). Fractions eluted with 0.2-0.4M NaCl were collected and furtherpurified on a 40 ml Phenyl Sepharose 6 Fast Flow column (GE 17-0965-05)with a linear (NH₄)₂SO₄ gradient (1.2-0 M). Fractions from the columnwere analyzed by SDS-PAGE using a NUPAGE® NOVEX® Bis-Tris Gel, 1.5MM15W.Fractions containing a band at approximately 80 kDa were pooled andconcentrated by ultrafiltration.

Example 7 Rhizomucor pusillus Genomic DNA Extraction

Rhizomucor pusillus strain NN046782 was inoculated onto a PDA plate andincubated for 3 days at 45° C. in the darkness. Several mycelia-PDAplugs were inoculated into 500 ml shake flasks containing 100 ml of FG4medium. The flasks were incubated for 3 days at 45° C. with shaking at160 rpm. The mycelia were collected by filtration through MIRACLOTH®(Calbiochem, La Jolla, Calif., USA) and frozen under liquid nitrogen.Frozen mycelia were ground, by a mortar and a pestle, to a fine powder,and genomic DNA was isolated using DNeasy® Plant Maxi Kit (QIAGEN Inc.,Valencia, Calif., USA) following the manufacturer's instruction.

Example 8 Genome Sequencing, Assembly and Annotation

The extracted genomic DNA samples were delivered to Beijing GenomeInstitute (BGI, Shenzhen, China) for genome sequencing using anILLUMINA® GA2 System (Illumina, Inc., San Diego, Calif., USA). The rawreads were assembled at BGI using program SOAPdenovo (Li et al., 2010,Genome Research 20(2): 265-72). The assembled sequences were analyzedusing standard bioinformatics methods for gene identification andfunctional prediction. GenelD (Parra et al., 2000, Genome Research10(4):511-515) was used for gene prediction. Blastall version 2.2.10(Altschul et al, 1990, J. Mol. Biol. 215 (3): 403-410, National Centerfor Biotechnology Information (NCBI), Bethesda, Md., USA) and HMMERversion 2.1.1 (National Center for Biotechnology Information (NCBI),Bethesda, Md., USA) were used to predict function based on structuralhomology. The catalases were identified by analysis of the Blastresults. The Agene program (Munch and Krogh, 2006, BMC Bioinformatics 7:263) and SignalP program (Nielsen et al., 1997, Protein Engineering 10:1-6) were used to identify starting codons. The SignalP program wasfurther used to predict the signal peptide. Pepstats (Rice et al., 2000,Trends Genet. 16(6): 276-277) was used to predict isoelectric points andmolecular weights of the deduced amino acid sequences.

Example 9 Cloning of the Rhizomucor pusillus Catalase Genes from GenomicDNA

Two catalase genes, shown in Table 2, were selected for expression.

TABLE 2 catalase genes Gene name DNA sequence Protein sequencecat_ZY654893_6661 SEQ ID NO: 3 SEQ ID NO: 4 cat_ZY654878_5541 SEQ ID NO:5 SEQ ID NO: 6

Based on DNA information (SEQ ID NO: 3 and SEQ ID NO: 5) obtained fromgenome sequencing, oligonucleotide primers, shown below in Table 3, weredesigned to amplify the catalase genes from the genomic DNA ofRhizomucor pusillus NN046782. Primers were synthesized by Invitrogen,Beijing, China.

TABLE 3 primers SEQID3_forward ACACAACTGGGGATCC ACC SEQ ID NO: 11atgcgactaggtgccttggca SEQID3_reverse GTCACCCTCTAGATCT atcg SEQ ID NO: 12attgagttgtacaagttcagc tacagc SEQID5_forward ACACAACTGGGGATCC ACCSEQ ID NO: 13 atgaaagccggttcgcttctc SEQID5_reverse GTCACCCTCTAGATCT cataSEQ ID NO: 14 tacgtaggactgggatgataa ctgtg

Lowercase characters of the forward primer represent the coding regionsof the gene and lowercase characters of the reverse primer represent theflanking region of the gene, while capitalized parts were homologous tothe insertion sites of pPFJO355 (WO2011005867).

For cat_ZY654893 6661, 20 picomoles of primer pair (SEQID3_forward andSEQID3_reverse) were used in a PCR reaction composed of 2 μl ofRhizomucor pusillus NN046782 genomic DNA, 10 μl of 5×GC Buffer, 1.5 μlof DMSO, 2.5 mM each of dATP, dTTP, dGTP, and dCTP, and 0.6 unit ofPHUSION™ High-Fidelity DNA Polymerase (Finnzymes Oy, Espoo, Finland) ina final volume of 50 μl. The amplification was performed using a PeltierThermal Cycler (M J Research Inc., South San Francisco, Calif., USA)programmed for denaturing at 98° C. for 1 minute; 6 cycles of denaturingat 98° C. for 30 seconds, annealing at 65° C. for 30 seconds, with a 1°C. decrease per cycle and elongation at 72° C. for 2.5 minutes; andanother 25 cycles each at 94° C. for 30 seconds, 59° C. for 30 secondsand 72° C. for 2.5 minutes; and a final extension at 72° C. for 5minutes. The heat block then went to a 4° C. soak cycle.

For cat_ZY654893 5541, 20 picomoles of primer pair (SEQID5_forward andSEQID5_reverse) were used in a PCR reaction composed of 2 μl ofRhizomucor pusillus NN046782 genomic DNA, 5 μl of 10×HIFI Buffer, 2 μlof 50 mM MgSO4, 2.5 mM each of dATP, dTTP, dGTP, and dCTP, and 2.5 unitsof PLATINUM® Taq DNA Polymerase High Fidelity (Invitrogen Corporation,Carlsbad, Calif., USA) in a final volume of 50 μl. The amplification wasperformed using a Peltier Thermal Cycler (M J Research Inc., South SanFrancisco, Calif., USA) programmed for denaturing at 94° C. for 1minute; 6 cycles of denaturing at 94° C. for 15 seconds, annealing at60° C. for 30 seconds, with a 1° C. decrease per cycle and elongation at68° C. for 3 minutes; and another 23 cycles each at 94° C. for 15seconds, 58° C. for 30 seconds and 68° C. for 3 minutes; and a finalextension at 68° C. for 5 minutes. The heat block then went to a 4° C.soak cycle.

The PCR products were isolated by 1.0% agarose gel electrophoresis using90 mM Tris-borate and 1 mM EDTA (TBE) buffer where a single product bandof each reaction around the expected size, ˜2.6 kb and ˜2.8 kb forcat_ZY654893_6661 and cat_ZY654893_5541 respectively, was visualizedunder UV light. PCR products were then purified from solution by usingan ILLUSTRA® GFX® PCR DNA and Gel Band Purification Kit (GE Healthcare,Buckinghamshire, UK) according to the manufacturer's instructions.

Plasmid pPFJO355 was digested with Bam HI and Bgl II, isolated by 1.0%agarose gel electrophoresis using TBE buffer, and purified using anILLUSTRA® GFX® PCR DNA and Gel Band Purification Kit according to themanufacturer's instructions.

An IN-FUSION™ CF Dry-down Cloning Kit (Clontech Laboratories, Inc.,Mountain View, Calif., USA) was used to clone the fragment directly intothe expression vector pPFJO355, without the need for restrictiondigestion and ligation.

TABLE 4 plasmids Gene name Plasmid DNA map cat_ZY654893_6661pCat_ZY654893_6661 FIG. 6 cat_ZY654878_5541 pCat_ZY654878_5541 FIG. 7

The PCR products and the digested vector were ligated together using anIN-FUSION™ CF Dry-down PCR Cloning resulting in plasmids shown in Table4: pCat_ZY654893_6661 (FIG. 6) and pCat_ZY654878_5541 (FIG. 7), in whichthe transcription of Ruzomucor pusillus catalase genes was under thecontrol of a promoter from the gene for Aspergillus orzyaealpha-amylase. The cloning operation was conducted according to themanufacturer's instruction. In brief, 30 ng of pPFJO355 digested withBam HI and Bgl II, and 60 ng of the purified Ruzomucor pusillus catalasePCR products were added to the reaction vial and resuspended the powderin a final volume of 10 μl with addition of deionized water. Thereaction product was incubated at 37° C. for 15 minutes and then 50° C.for 15 minutes. Three microlitres of the reaction were used to transformE. coli TOP10 competent cells (TIANGEN Biotech (Beijing) Co. Ltd.,Beijing, China). E. coli transformants containing expression constructswere detected by colony PCR and plasmid DNA was prepared using aQIAprep® Spin Miniprep Kit (QIAGEN GmbH, Hilden, Germany). The Ruzomucorpusillus catalase genes inserted in pCat_ZY654893_6661 andpCat_ZY654878_5541 was confirmed by DNA sequencing using a 3730XL DNAAnalyzer (Applied Biosystems Inc, Foster City, Calif., USA).

Example 10 Penicillium emersonii Genomic DNA Extraction

Penicillium emersonii strain NN051602 was inoculated onto a PDA plateand incubated for 3 days at 45° C. in the darkness. Several mycelia-PDAplugs were inoculated into 500 ml shake flasks containing 100 ml of YPGmedium. The flasks were incubated for 3 days at 45° C. with shaking at160 rpm. The mycelia were collected by filtration through MIRACLOTH®(Calbiochem, La Jolla, Calif., USA) and frozen under liquid nitrogen.Frozen mycelia were ground, by a mortar and a pestle, to a fine powder,and genomic DNA was isolated using a Large-Scale Column Fungal DNAout(Baoman Biotechnology, Shanghai, China) according to the manufacturer'sinstructions.

Example 11 Genome Sequencing, Assembly and Annotation

The extracted genomic DNA samples were delivered to Beijing GenomeInstitute (BGI, Shenzhen, China) for genome sequencing using anILLUMINA® GA2 System (Illumina, Inc., San Diego, Calif., USA). The rawreads were assembled at BGI using program SOAPdenovo (Li et al., 2010,Genome Research 20(2): 265-72). The assembled sequences were analyzedusing standard bioinformatics methods for gene identification andfunctional prediction. GenelD (Parra et al., 2000, Genome Research10(4):511-515) was used for gene prediction. Blastall version 2.2.10(Altschul et al., 1990, J. Mol. Biol. 215 (3): 403-410, National Centerfor Biotechnology Information (NCBI), Bethesda, Md., USA) and HMMERversion 2.1.1 (National Center for Biotechnology Information (NCBI),Bethesda, Md., USA) were used to predict function based on structuralhomology. The catalase was identified by analysis of the Blast results.The Agene program (Munch and Krogh, 2006, BMC Bioinformatics 7: 263) andSignalP program (Nielsen et al., 1997, Protein Engineering 10: 1-6) wereused to identify start codons. The SignalP program was further used topredict signal peptide. Pepstats (Rice et al., 2000, Trends Genet.16(6): 276-277) was used to predict isoelectric point of proteins, andmolecular weight of the deduced amino acid sequence.

Example 12 Cloning of the Penicillium emersonii Catalase from GenomicDNA

One catalase gene, PE04230007241 (SEQ ID NO: 7), was selected forexpression cloning.

Based on the gene information (SEQ ID NO: 7) obtained by genomesequencing, oligonucleotide primers, shown below in Table 5, weredesigned to amplify the catalase gene, PE04230007241, from the genomicDNA of Penicillium emersonii. Primers were synthesized by

Invitrogen, Beijing, China.

TABLE 5 primers Forward primer 5′ ACACAACTGGGGATCC SEQ ID NO: 15ACC atgcgcgcagtgcag ct 3′ Reverse primer 5′ GTCACCCTCTAGATCTSEQ ID NO: 16 gtcgactattccaaccttcct atatggacac 3′

Lowercase characters of the forward primer represent the coding regionsof the gene and lowercase characters of the reverse primer represent theflanking region of the gene, while capitalized parts were homologous tothe insertion sites of pPFJO355 vector which has been described inWO2011005867.

An IN-FUSION™ CF Dry-down Cloning Kit (Clontech Laboratories, Inc.,Mountain View, Calif., USA) was used to clone the fragment directly intothe expression vector pPFJO355, without the need for restrictiondigestion and ligation.

Twenty picomoles of each forward and reverse primer pair were used in aPCR reaction composed of 2 μl of Penicillium emersonii genomic DNA, 10μl of 5×GC Buffer, 1.5 μl of DMSO, 2.5 mM each of dATP, dTTP, dGTP, anddCTP, and 0.6 unit of PHUSION™ High-Fidelity DNA Polymerase (FinnzymesOy, Espoo, Finland) in a final volume of 50 μl. The amplification wasperformed using a Peltier Thermal Cycler (M J Research Inc., South SanFrancisco, Calif., USA) programmed for denaturing at 98° C. for 1minute; 8 cycles of denaturing at 98° C. for 15 seconds, annealing at65° C. for 30 seconds, with a 1° C. decrease per cycle and elongation at72° C. for 3 minute 15 second; and another 22 cycles each at 98° C. for15 seconds, 58 C for 30 seconds and 72° C. for 3 minute 15 second; and afinal extension at 72° C. for 10 minutes. The heat block then went to a4° C. soak cycle.

The reaction product was isolated by 1.0% agarose gel electrophoresisusing 90 mM Tris-borate and 1 mM EDTA (TBE) buffer where a ˜2.5 kbproduct band was excised from the gel, and purified using an ILLUSTRA®GFX® PCR DNA and Gel Band Purification Kit (GE Healthcare,Buckinghamshire, UK) according to the manufacturer's instructions.

Plasmid pPFJO355 was digested with Bam HI and Bgl II, isolated by 1.0%agarose gel electrophoresis using TBE buffer, and purified using anILLUSTRA® GFX® PCR DNA and Gel Band Purification Kit according to themanufacturer's instructions.

The PCR product and the digested vector were ligated together using anIN-FUSION™ CF Dry-down PCR Cloning kit resulting in pCat_PE04230007241(FIG. 8) in which the transcription of the Penicillium emersoniicatalase gene was under the control of a promoter from the gene forAspergillus oryzae alpha-amylase. The cloning operation was conductedaccording to the manufacturer's instruction. In brief, 30 ng of pPFJO355digested with Bam HI and Bgl II, and 60 ng of the purified Penicilliumemersonii catalase gene PCR product were added to the reaction vials andresuspended the powder in a final volume of 10 μl with addition ofdeionized water. The reaction was incubated at 37° C. for 15 minutes andthen 50° C. for 15 minutes. Three microlitres of the reaction were usedto transform E. coli TOP10 competent cells (TIANGEN Biotech (Beijing)Co. Ltd., Beijing, China). An E. coli transformant containingpCat_PE04230007241 was detected by colony PCR. Colony PCR is a methodfor quick screening of plasmid inserts directly from E. coli colonies.Briefly, in the premixed PCR solution aliquot in each PCR tube,including PCR buffer, MgCl₂, dNTPs, and primer pairs from which the PCRfragment was generated, a single colony was added by picking with asterile tip and twirling the tip in the reaction solution. Normally 7-10colonies were screened. After the PCR, reactions were analyzed by 1.0%agarose gel electrophoresis using TBE buffer. Plasmid DNA was preparedfrom colonies showing inserts with the expected sizes using a QIAprep®Spin Miniprep Kit. The Penicillium emersonii catalase gene inserted inpCat_PE04230007241 was confirmed by DNA sequencing using a 3730XL DNAAnalyzer (Applied Biosystems Inc, Foster City, Calif., USA).

Example 13 Expression of Penicillium emersonii Catalase Gene inAspergillus oryzae

Aspergillus oryzae HowB101 (described in patent WO9535385 example 1)protoplasts were prepared according to the method of Christensen et al,1988, Bio/Technology 6: 1419-1422 and transformed with 3 μg ofpCat_PE04230007241.

The transformation of Aspergillus oryzae HowB101 with pCat_PE04230007241yielded about 50 transformants. Four transformants were isolated toindividual Minimal medium plates.

Four transformants were inoculated separately into 3 ml of YPM medium ina 24-well plate and incubated at 30° C., with mixing at 150 rpm. After 3days incubation, 20 μl of supernatant from each culture were analyzed bySDS-PAGE using a NUPAGE® NOVEX® 4-12% Bis-Tris Gel with 50 mM MES(Invitrogen Corporation, Carlsbad, Calif., USA) according to themanufacturer's instructions. The resulting gel was stained withINSTANTBLUE™ (Expedeon Ltd., Babraham Cambridge, UK). SDS-PAGE profilesof the cultures showed that all transformants had a band ofapproximately 80 kDa. The expression strain was designated as O6YTS.

Example 14 Fermentation of Aspergillus oryzae Expression Strain O6YTS

A slant of the expression strain, O6YTS, was washed with 10 ml of YPMmedium and inoculated into 7 2-liter flasks, each containing 400 ml ofYPM medium to generate broth. The culture was harvested on day 3 andfiltered using a 0.45 μm DURAPORE® Membrane (Millipore, Bedford, Mass.,USA).

Example 15 Purification of Recombinant Penicillium emersonii Catalasefrom Aspergillus Oryzae O6YTS

2800 ml volume of filtered supernatant of the recombinant strain O6YTS(Example 14) was precipitated with ammonium sulfate (80% saturation),re-dissolved in 50 ml 20 mM Tris-HCl buffer, pH8.0, dialyzed against thesame buffer, and filtered through a 0.45 μm filter. The final volume was80 ml. The solution was applied to a 40 ml Q SEPHAROSE® Fast Flow column(GE Healthcare, Buckinghamshire, UK) equilibrated in 20 mM Tris-HClbuffer, pH8.0. Fractions eluted with 0.18-0.25M NaCl were analyzed bySDS-PAGE using a NUPAGE® NOVEX® 4-12% Bis-Tris Gel, 1.5MM15W. Fractionscontaining a band at approximately 80 kDa were pooled and concentratedby ultrafiltration.

Example 16 Characterization of the genomic DNAs encoding catalases

The genomic DNA sequence (SEQ ID NO: 1) and deduced amino acid sequence(SEQ ID NO: 2) of the Malbranchea cinnamomea catalase coding sequence isshown in FIG. 5. The coding sequence is 2622 bp including the stop codonand is interrupted by 6 introns of 81 bp (nucleotides 289 to 369), 69 bp(nucleotides 404 to 472), 72 bp (nucleotides 665 to 736), 68 bp(nucleotides 1153 to 1220), 71 bp (nucleotides 1386 to 1456) and 71 bp(nucleotides 1632 to 1702). The G+C content of the mature polypeptidecoding sequence without introns and stop codon is 52.8%. The encodedpredicted protein is 729 amino acids. Using the SignalP program (Nielsenet al., 1997, Protein Engineering 10: 1-6), a signal peptide of 16residues was predicted. The predicted mature protein contains 713 aminoacids with a predicted molecular weight of 79259.08 Dalton and predictedisoelectric point of 5.13. The catalase catalytic domain was predictedto be amino acids 17 to 723, by aligning the amino acid sequence usingBLAST, where the single most significant alignment within a subfamilywas used to predict the catalytic domain.

A comparative pairwise global alignment of amino acid sequences wasdetermined using the Needleman and Wunsch algorithm (Needleman andWunsch, 1970, J. Mol. Biol. 48: 443-453) with gap open penalty of 10,gap extension penalty of 0.5, and the EBLOSUM62 matrix. The alignmentshowed that the mature part of amino acid sequence of the Malbrancheacinnamomea coding sequence encoding the catalase polypeptide shares75.36% identity to the deduced amino acid sequence of catalase fromNeosartorya fischeri (accession number UNIPROT:A1 DJU9).

The genomic DNA sequence (SEQ ID NO: 3) and deduced amino acid sequence(SEQ ID NO: 4) of the Rhizomucor pusillus catalase coding sequence isshown in FIG. 6. The coding sequence is 2711 bp including the stop codonand is interrupted by 7 introns of 124 bp (nucleotides 291 to 414), 64bp (nucleotides 648 to 711), 77 bp (nucleotides 745 to 821), 67 bp(nucleotides 1021 to 1087), 58 bp (nucleotides 1179 to 1236), 70 bp(nucleotides 1520 to 1589) and 58 bp (nucleotides 1729 to 1786). The G+Ccontent of the mature polypeptide coding sequence without introns andstop codon is 50%. The encoded predicted protein is 730 amino acids.Using the SignalP program (Nielsen et al., 1997, Protein Engineering 10:1-6), a signal peptide of 20 residues was predicted. The predictedmature protein contains 710 amino acids with a predicted molecularweight of 79485.02 Dalton and predicted isoelectric point of 6.03. Thecatalase catalytic domain was predicted to be amino acids 38 to 723, byaligning the amino acid sequence using BLAST, where the single mostsignificant alignment within a subfamily was used to predict thecatalytic domain.

A comparative pairwise global alignment of amino acid sequences wasdetermined using the Needleman and Wunsch algorithm (Needleman andWunsch, 1970, J. Mol. Biol. 48: 443-453) with gap open penalty of 10,gap extension penalty of 0.5, and the EBLOSUM62 matrix. The alignmentshowed that the mature part of amino acid sequence of the Rhizomucorpusillus coding sequence encoding the catalase polypeptide shares 53.67%identity to the catalase from Bacillus pseudofirmus (accession numberUNIPROT:P30266).

The genomic DNA sequence (SEQ ID NO: 5) and deduced amino acid sequence(SEQ ID NO: 6) of the Rhizomucor pusillus catalase coding sequence isshown in FIG. 7. The coding sequence is 2673 bp including the stop codonand is interrupted by 7 introns of 72 bp (nucleotides 284 to 355), 100bp (nucleotides 584 to 683), 75 bp (nucleotides 717 to 791), 57 bp(nucleotides 991 to 1047), 72 bp (nucleotides 1133 to 1204) and 72 bp(nucleotides 1259 to 1330) and 71 bp (nucleotides 1697 to 1767). The G+Ccontent of the mature polypeptide coding sequence without introns andstop codon is 50.3%. The encoded predicted protein is 717 amino acids.Using the SignalP program (Nielsen et al., 1997, Protein Engineering 10:1-6), a signal peptide of 24 residues was predicted. The predictedmature protein contains 693 amino acids with a predicted molecularweight of 77995.99 Dalton and predicted isoelectric point of 5.66. Thecatalase catalytic domain was predicted to be amino acids 38 to 711, byaligning the amino acid sequence using BLAST, where the single mostsignificant alignment within a subfamily was used to predict thecatalytic domain.

A comparative pairwise global alignment of amino acid sequences wasdetermined using the Needleman and Wunsch algorithm (Needleman andWunsch, 1970, J. Mol. Biol. 48: 443-453) with gap open penalty of 10,gap extension penalty of 0.5, and the EBLOSUM62 matrix. The alignmentshowed that the mature part of amino acid sequence of the Malbrancheacinnamomea coding sequence encoding the catalase polypeptide shares55.94% identity to the catalase from Bacillus subtilis (accession numberUNIPROT:P42234).

The genomic DNA sequence (SEQ ID NO: 7) and deduced amino acid sequence(SEQ ID NO: 8) of the Penicillium emersonii catalase coding sequence isshown in FIG. 8. The coding sequence is 2479 bp including the stop codonand is interrupted by 5 introns of 53 bp (nucleotides 328 to 380), 50 bp(nucleotides 607 to 656), 51 bp (nucleotides 1073 to 1123), 52 bp(nucleotides 1289 to 1340), and 47 bp (nucleotides 1516 to 1562). TheG+C content of the mature polypeptide coding sequence without intronsand stop codon is 58.6%. The encoded predicted protein is 741 aminoacids. Using the SignalP program (Nielsen et al., 1997, ProteinEngineering 10: 1-6), a signal peptide of 19 residues was predicted. Thepredicted mature protein contains 722 amino acids with a predictedmolecular weight of 79578.21 Dalton and predicted isoelectric point of5.12. The catalase catalytic domain was predicted to be amino acids 20to 740, by aligning the amino acid sequence using BLAST, where thesingle most significant alignment within a subfamily was used to predictthe catalytic domain.

A comparative pairwise global alignment of amino acid sequences wasdetermined using the Needleman and Wunsch algorithm (Needleman andWunsch, 1970, J. Mol. Biol. 48: 443-453) with gap open penalty of 10,gap extension penalty of 0.5, and the EBLOSUM62 matrix. The alignmentshowed that the mature part of amino acid sequence of the Penicilliumemersonii coding sequence encoding the catalase polypeptide shares82.38% identity to a catalase from Thermoascus aurantiacus (SEQ ID NO: 4in JP2007143405-A)

Example 17 Catalase Activity Determination

Catalase activity was detected using 3% hydrogen peroxide as substrate.3% hydrogen peroxide was prepared by 10 times dilution of 30% hydrogenperoxide with double distilled H₂O (ddH₂O). 50 μl of 3% hydrogenperoxide was added to a well of the microtiter plate. Then 20 μl ofpurified catalase sample was added to the same well. The reaction waskept at room temperature for 10-30 seconds. The catalase activity wasdetermined by observation of bubble (oxygen) generation.

As bubble (oxygen) generation was observed, Malbranchea cinnamomeacatalase (Example 6) sample showed catalase activity and Penicilliumemersonii catalase (Example 15) sample showed catalase activity.

Example 18 Catalase Activity Assay

The purified Penicillium emersonii catalase (Example 15) was checked forcatalase activity by using the following protocol.

The substrate was prepared by 1000 times dilution of 30% H₂O₂ (fromXilong Chemical, Guangdong, China) with double distilled H₂O (ddH₂O),the final concentration was 10.3 mM. The reaction was started by adding1 μl of purified Penicillium emersonii catalase sample into 1000 μl ofsubstrate. The optical density (OD) at 240 nm was read by Ultrospec 3300(GE Healthcare, Buckinghamshire, UK) at second of 0 and 16 respectively,and the decrease of the OD (from 0.505 to 0.284) showed the relativeactivity of the Penicillium emersonii catalase.

Example 19 Boosting Effect of Penicillium emersonii Catalase onHydrolysis of PCS

Corn stover was pretreated at the U.S. Department of Energy NationalRenewable Energy Laboratory (NREL) using dilute sulfuric acid atconditions of 190° C., 1 minute residence time, 0.05 g acid/g drybiomass, and at a 30% total solid concentration in a pretreatmentreactor. Pretreated corn stover (PCS) was hydrolyzed at an initial totalsolid (TS) of 10% and total weight of hydrolysis system of 20 g.Trichoderma reesei cellulase composition (CELLIC® CTec2 available fromNovozymes A/S, Bagsvaerd, Denmark) was added into the PCS for enzymatichydrolysis. Five percent by weight of Trichoderma reesei cellulasecomposition was replaced with P. emersonii catalase based on proteinamount and the total enzyme dose was 4 mg/g cellulose. The hydrolysissystem with Trichoderma reesei cellulase composition but withoutcatalase was used as a control. The flasks were incubated at 50° C. for72 hours, with shaking at 130 rpm. After hydrolysis was completed, thesugar was analyzed by High Performance Liquid Chromatography (HPLC).

For HPLC measurement, the collected samples were filtered using 0.22 μmsyringe filters (Millipore, Bedford, Mass., USA) and the filtrates wereanalyzed for sugar content as described below. The sugar concentrationsof samples diluted in 0.005 M H₂SO₄ were measured using a 7.8×300 mmAMINEX® HPX-87H column (Bio-Rad Laboratories, Inc., Hercules, Calif.,USA) by elution with 0.005 M H₂SO₄ at 65° C. at a flow rate of 0.7 mlper minute, and quantification by integration of the glucose signal fromrefractive index detection (CHEMSTATION®, AGILENT® 1100 HPLC, AgilentTechnologies, Santa Clara, Calif., USA) calibrated by pure sugarsamples. The resultant glucose was used to calculate the percentage ofglucose yield from glucans for each reaction. Measured sugarconcentrations were adjusted for the appropriate dilution factor. Thenet concentrations of enzymatically-produced sugars were determined byadjusting the measured sugar concentrations for corresponding backgroundsugar concentrations in unwashed biomass at zero time point. All HPLCdata processing was performed using MICROSOFT EXCEL™ software(Microsoft, Richland, Wash., USA).

The degree of glucose conversion to glucose was calculated according tothe publication by Zhu, Y., et al. 2010, Bioresource Technology. 102(3):2897-2903.

The results as shown in Table 6 demonstrated that PCS conversion toglucose can be improved significantly by adding small amounts ofcatalase.

TABLE 6 Effect of catalase from P. emersonii on glucose conversion ofPCS. Control P. emersonii Catalase Glucose conversion (%) 48.6 ± 0.754.3 ± 0.8

The present invention is further described by the following numberedparagraphs:

[1] An isolated polypeptide having catalase activity, selected from thegroup consisting of:

(a) a polypeptides having at least 83%, e.g., at least 84%, at least85%, at least 86%, at least 87%, at least 88%, at least 89%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to the mature polypeptide of SEQ ID NO: 8, apolypeptide having at least 76%, e.g., at least 77%, at least 78%, atleast 79%, at least 80%, at least 81%, at least 82%, at least 83%, atleast 84%, at least 85%, at least 86%, at least 87%, at least 88%, atleast 89%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or 100% sequence identity to the mature polypeptide of SEQ IDNO: 2, a polypeptides having at least 60%, e.g., at least 65%, at least70%, at least 75%, at least 78%, at least 80%, at least 81%, at least82%, at least 83%, at least 84%, at least 85%, at least 86%, at least87%, at least 88%, at least 89%, at least 90%, at least 91%, at least92%, at least 93%, at least 94%, at least 95%, at least 96%, at least97%, at least 98%, at least 99%, or 100% sequence identity to the maturepolypeptide of SEQ ID NO: 4, or a polypeptides having at least 60%,e.g., at least 65%, at least 70%, at least 75%, at least 78%, at least80%, at least 81%, at least 82%, at least 83%, at least 84%, at least85%, at least 86%, at least 87%, at least 88%, at least 89%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to the mature polypeptide of SEQ ID NO: 6;

(b) a polypeptide encoded by a polynucleotide that hybridizes under lowstringency conditions, medium stringency conditions, medium-highstringency conditions, high stringency conditions, or very highstringency conditions with (i) the mature polypeptide coding sequence ofSEQ ID NO: 7, the mature polypeptide coding sequence of SEQ ID NO: 1,the mature polypeptide coding sequence of SEQ ID NO: 3, or the maturepolypeptide coding sequence of SEQ ID NO: 5, (ii) the cDNA sequencethereof, or (iii) the full-length complement of (i) or (ii);

(c) a polypeptide encoded by a polynucleotide having at least 83%, e.g.,at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, atleast 89%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or 100% sequence identity to the mature polypeptide codingsequence of SEQ ID NO: 7; or the cDNA sequence thereof, a polypeptideencoded by a polynucleotide having at least 76%, e.g., at least 77%, atleast 78%, at least 79%, at least 80%, at least 81%, at least 82%, atleast 83%, at least 84%, at least 85%, at least 86%, at least 87%, atleast 88%, at least 89%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100% sequence identity to the maturepolypeptide coding sequence of SEQ ID NO: 1, a polypeptide encoded by apolynucleotide having at least 60%, e.g., at least 65%, at least 70%, atleast 75%, at least 78%, at least 80%, at least 81%, at least 82%, atleast 83%, at least 84%, at least 85%, at least 86%, at least 87%, atleast 88%, at least 89%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100% sequence identity to the maturepolypeptide coding sequence of SEQ ID NO: 3, or a polypeptide encoded bya polynucleotide having at least 60%, e.g., at least 65%, at least 70%,at least 75%, at least 78%, at least 80%, at least 81%, at least 82%, atleast 83%, at least 84%, at least 85%, at least 86%, at least 87%, atleast 88%, at least 89%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100% sequence identity to the maturepolypeptide coding sequence of SEQ ID NO: 5;

(d) a variant of the mature polypeptide of SEQ ID NO: 8, a variant ofthe mature polypeptide of SEQ ID NO: 2, a variant of the maturepolypeptide of SEQ ID NO: 4, or a variant of the mature polypeptide ofSEQ ID NO: 6, comprising a substitution, deletion, and/or insertion atone or more (e.g., several) positions; and

(e) a fragment of the polypeptide of (a), (b), (c), or (d) that hascatalase activity.

[2] The polypeptide of paragraph 1, having at least 83%, e.g., at least84%, at least 85%, at least 86%, at least 87%, at least 88%, at least89%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, at least99%, or 100% sequence identity to the mature polypeptide of SEQ ID NO:8, having at least 76%, e.g., at least 77%, at least 78%, at least 79%,at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, atleast 85%, at least 86%, at least 87%, at least 88%, at least 89%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% sequence identity to the mature polypeptide of SEQ ID NO: 2, havingat least 60%, e.g., at least 65%, at least 70%, at least 75%, at least78%, at least 80%, at least 81%, at least 82%, at least 83%, at least84%, at least 85%, at least 86%, at least 87%, at least 88%, at least89%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, at least99%, or 100% sequence identity to the mature polypeptide of SEQ ID NO:4, or having at least 60%, e.g., at least 65%, at least 70%, at least75%, at least 78%, at least 80%, at least 81%, at least 82%, at least83%, at least 84%, at least 85%, at least 86%, at least 87%, at least88%, at least 89%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, at least 99%, or 100% sequence identity to the mature polypeptideof SEQ ID NO: 6.

[3] The polypeptide of paragraph 1 or 2, which is encoded by apolynucleotide that hybridizes under low stringency conditions,low-medium stringency conditions, medium stringency conditions,medium-high stringency conditions, high stringency conditions, or veryhigh stringency conditions with (i) the mature polypeptide codingsequence of SEQ ID NO: 7, the mature polypeptide coding sequence of SEQID NO: 1, the mature polypeptide coding sequence of SEQ ID NO: 3, or themature polypeptide coding sequence of SEQ ID NO: 5, (ii) the cDNAsequence thereof, or (iii) the full-length complement of (i) or (ii).[4] The polypeptide of any of paragraphs 1-3, which is encoded by apolynucleotide having at least 83%, e.g., at least 84%, at least 85%, atleast 86%, at least 87%, at least 88%, at least 89%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100% sequenceidentity to the mature polypeptide coding sequence of SEQ ID NO: 7, orthe cDNA sequence thereof, which is encoded by a polynucleotide havingat least 76%, e.g., at least 77%, at least 78%, at least 79%, at least80%, at least 81%, at least 82%, at least 83%, at least 84%, at least85%, at least 86%, at least 87%, at least 88%, at least 89%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to the mature polypeptide coding sequence of SEQ IDNO: 1, or the cDNA sequence thereof, which is encoded by apolynucleotide having at least 60%, e.g., at least 65%, at least 70%, atleast 75%, at least 78%, at least 80%, at least 81%, at least 82%, atleast 83%, at least 84%, at least 85%, at least 86%, at least 87%, atleast 88%, at least 89%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100% sequence identity to the maturepolypeptide coding sequence of SEQ ID NO: 3, or the cDNA sequencethereof, or which is encoded by a polynucleotide having at least 60%,e.g., at least 65%, at least 70%, at least 75%, at least 78%, at least80%, at least 81%, at least 82%, at least 83%, at least 84%, at least85%, at least 86%, at least 87%, at least 88%, at least 89%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to the mature polypeptide coding sequence of SEQ IDNO: 5, or the cDNA sequence thereof.

[5] The polypeptide of any of paragraphs 1-4, comprising or consistingof SEQ ID NO: 8, SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6, or themature polypeptide of SEQ ID NO: 8, the mature polypeptide of SEQ ID NO:2, the mature polypeptide of SEQ ID NO: 4, or the mature polypeptide ofSEQ ID NO: 6.

[6] The polypeptide of paragraph 5, wherein the mature polypeptide isamino acids 20 to 741 of SEQ ID NO: 8, amino acids 17 to 729 of SEQ IDNO: 2, amino acids 21 to 730 of SEQ ID NO: 4, or amino acids 25 to 717of SEQ ID NO: 6.

[7] The polypeptide of any of paragraphs 1-4, which is a variant of themature polypeptide of SEQ ID NO: 8, a variant of the mature polypeptideof SEQ ID NO: 2, a variant of the mature polypeptide of SEQ ID NO: 4, ora variant of the mature polypeptide of SEQ ID NO: 6, comprising asubstitution, deletion, and/or insertion at one or more (e.g., several)positions. [8] The polypeptide of paragraph 1, which is a fragment ofSEQ ID NO: 8, SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6, wherein thefragment has catalase activity.

[9] An isolated polypeptide comprising a catalytic domain selected fromthe group consisting of:

(a) a catalytic domain having at least 83%, e.g., at least 84%, at least85%, at least 86%, at least 87%, at least 88%, at least 89%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to amino acids 20 to 740 of SEQ ID NO: 8; a catalyticdomain having at least 76%, e.g., at least 77%, at least 78%, at least79%, at least 80%, at least 81%, at least 82%, at least 83%, at least84%, at least 85%, at least 86%, at least 87%, at least 88%, at least89%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, at least99%, or 100% sequence identity to amino acids 17 to 723 of SEQ ID NO: 2;a catalytic domain having at least 60%, e.g., at least 65%, at least70%, at least 75%, at least 78%, at least 80%, at least 81%, at least82%, at least 83%, at least 84%, at least 85%, at least 86%, at least87%, at least 88%, at least 89%, at least 90%, at least 91%, at least92%, at least 93%, at least 94%, at least 95%, at least 96%, at least97%, at least 98%, at least 99%, or 100% sequence identity to aminoacids 38 to 723 of SEQ ID NO: 4; or a catalytic domain having at least60%, e.g., at least 65%, at least 70%, at least 75%, at least 78%, atleast 80%, at least 81%, at least 82%, at least 83%, at least 84%, atleast 85%, at least 86%, at least 87%, at least 88%, at least 89%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% sequence identity to amino acids 38 to 711 of SEQ ID NO: 6;

(b) a polypeptide encoded by a polynucleotide that hybridizes under low,medium, medium-high, high, or very high stringency conditions with (i)nucleotides 58 to 2473 of SEQ ID NO: 7, nucleotides 49 to 2601 of SEQ IDNO: 1, nucleotides 112 to 2687 of SEQ ID NO: 3, or nucleotides 112 to2652 of SEQ ID NO: 5, (ii) the cDNA sequence thereof, or (iii) thefull-length complement of (i) or (ii);

(c) a catalytic domain encoded by a polynucleotide having at least 83%,e.g., at least 84%, at least 85%, at least 86%, at least 87%, at least88%, at least 89%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, at least 99%, or 100% sequence identity to nucleotides 58 to 2473of SEQ ID NO: 7; a catalytic domain encoded by a polynucleotide havingat least 76%, e.g., at least 77%, at least 78%, at least 79%, at least80%, at least 81%, at least 82%, at least 83%, at least 84%, at least85%, at least 86%, at least 87%, at least 88%, at least 89%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to nucleotides 49 to 2601 of SEQ ID NO: 1; a catalyticdomain encoded by a polynucleotide having at least 60%, e.g., at least65%, at least 70%, at least 75%, at least 78%, at least 80%, at least81%, at least 82%, at least 83%, at least 84%, at least 85%, at least86%, at least 87%, at least 88%, at least 89%, at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, at least 99%, or 100% sequence identityto nucleotides 112 to 2687 of SEQ ID NO: 3; or a catalytic domainencoded by a polynucleotide having at least 60%, e.g., at least 65%, atleast 70%, at least 75%, at least 78%, at least 80%, at least 81%, atleast 82%, at least 83%, at least 84%, at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100% sequence identity tonucleotides 112 to 2652 of SEQ ID NO: 5;

(d) a variant of amino acids 20 to 740 of SEQ ID NO: 8, a variant ofamino acids 17 to 723 of SEQ ID NO: 2, a variant of amino acids 38 to723 of SEQ ID NO: 4, or a variant of amino acids 38 to 711 of SEQ ID NO:6, comprising a substitution, deletion, and/or insertion of one or more(e.g., several) positions; and

(e) a fragment of a catalytic domain of (a), (b), (c), or (e) which hascatalase activity.

[10] A composition comprising the polypeptide of any of paragraphs 1-9.

[11] An isolated polynucleotide encoding the polypeptide of any ofparagraphs 1-9.

[12] A nucleic acid construct or expression vector comprising thepolynucleotide of paragraph 11 operably linked to one or more (e.g.,several) control sequences that direct the production of the polypeptidein an expression host.

[13] A recombinant host cell comprising the polynucleotide of paragraph11 operably linked to one or more (e.g., several) control sequences thatdirect the production of the polypeptide.

[14] A method of producing the polypeptide of any of paragraphs 1-9,comprising:

(a) cultivating a cell, which in its wild-type form produces thepolypeptide, under conditions conducive for production of thepolypeptide; and optionally

(b) recovering the polypeptide.

[15] A method of producing a polypeptide having catalase activity,comprising:

(a) cultivating the host cell of paragraph 13 under conditions conducivefor production of the polypeptide; and optionally

(b) recovering the polypeptide.

[16] A transgenic plant, plant part or plant cell transformed with apolynucleotide encoding the polypeptide of any of paragraphs 1-9.

[17] A method of producing a polypeptide having catalase activity,comprising:

(a) cultivating the transgenic plant or plant cell of paragraph 16 underconditions conducive for production of the polypeptide; and

(b) recovering the polypeptide.

[18] A method of producing a mutant of a parent cell, comprisinginactivating a polynucleotide encoding the polypeptide of any ofparagraphs 1-9, which results in the mutant producing less of thepolypeptide than the parent cell.

[19] A mutant cell produced by the method of paragraph 18.

[20] The mutant cell of paragraph 19, further comprising a gene encodinga native or heterologous protein.

[21] A method of producing a protein, comprising:

(a) cultivating the mutant cell of paragraph 19 or 20 under conditionsconducive for production of the protein; and optionally

(b) recovering the protein.

[22] A double-stranded inhibitory RNA (dsRNA) molecule comprising asubsequence of the polynucleotide of paragraph 11, wherein optionallythe dsRNA is an siRNA or an miRNA molecule.

[23] The double-stranded inhibitory RNA (dsRNA) molecule of paragraph22, which is about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or moreduplex nucleotides in length.

[24] A method of inhibiting the expression of a polypeptide havingcatalase activity in a cell, comprising administering to the cell orexpressing in the cell the double-stranded inhibitory RNA (dsRNA)molecule of paragraph 22 or 23.

[25] A cell produced by the method of paragraph 24.

[26] The cell of paragraph 25, further comprising a gene encoding anative or heterologous protein.

[27] A method of producing a protein, comprising:

(a) cultivating the cell of paragraph 25 or 26 under conditionsconducive for production of the protein; and optionally

(b) recovering the protein.

[28] An isolated polynucleotide encoding a signal peptide comprising orconsisting of amino acids 1 to 19 of SEQ ID NO: 8, amino acids 1 to 16of SEQ ID NO: 2, amino acids 1 to 20 of SEQ ID NO: 4, or amino acids 1to 24 of SEQ ID NO: 6.

[29] A nucleic acid construct or expression vector comprising a geneencoding a protein operably linked to the polynucleotide of paragraph28, wherein the gene is foreign to the polynucleotide encoding thesignal peptide.

[30] A recombinant host cell comprising a gene encoding a proteinoperably linked to the polynucleotide of paragraph 28, wherein the geneis foreign to the polynucleotide encoding the signal peptide.

[31] A method of producing a protein, comprising:

(a) cultivating a recombinant host cell comprising a gene encoding aprotein operably linked to the polynucleotide of paragraph 28, whereinthe gene is foreign to the polynucleotide encoding the signal peptide,under conditions conducive for production of the protein; and optionally

(b) recovering the protein.

[32] A process for degrading or converting a cellulosic material,comprising: treating the cellulosic material with an enzyme compositionin the presence of the polypeptide having catalase activity of any ofparagraphs 1-9.

[33] The process of paragraph 32, wherein the cellulosic material ispretreated.

[34] The process of paragraph 32 or 33, wherein the enzyme compositioncomprises one or more (e.g., several) enzymes selected from the groupconsisting of a cellulase, a GH61 polypeptide having cellulolyticenhancing activity, a hemicellulase, an esterase, an expansin, alaccase, a ligninolytic enzyme, a pectinase, a peroxidase, a protease,and a swollenin.

[35] The process of paragraph 34, wherein the cellulase is one or more(e.g., several) enzymes selected from the group consisting of anendoglucanase, a cellobiohydrolase, and a beta-glucosidase.

[36] The process of paragraph 35, wherein the hemicellulase is one ormore (e.g., several) enzymes selected from the group consisting of axylanase, an acetylxylan esterase, a feruloyl esterase, anarabinofuranosidase, a xylosidase, and a glucuronidase.

[37] The process of any of paragraphs 32-36, further comprisingrecovering the degraded or converted cellulosic material.

[38] The process of paragraph 37, wherein the degraded cellulosicmaterial is a sugar.

[39] The process of paragraph 38, wherein the sugar is selected from thegroup consisting of glucose, xylose, mannose, galactose, and arabinose.

[40] A process for producing a fermentation product, comprising:

(a) saccharifying a cellulosic material with an enzyme composition inthe presence of the polypeptide having catalase activity of any ofparagraphs 1-9;

(b) fermenting the saccharified cellulosic material with one or more(e.g., several) fermenting microorganisms to produce the fermentationproduct; and optionally

(c) recovering the fermentation product from the fermentation.

[41] The process of paragraph 40, wherein the cellulosic material ispretreated.

[42] The process of paragraph 40 or 41, wherein the enzyme compositioncomprises the enzyme composition comprises one or more (e.g., several)enzymes selected from the group consisting of a cellulase, a GH61polypeptide having cellulolytic enhancing activity, a hemicellulase, anesterase, an expansin, a laccase, a ligninolytic enzyme, a pectinase, aperoxidase, a protease, and a swollenin.

[43] The process of paragraph 42, wherein the cellulase is one or more(e.g., several) enzymes selected from the group consisting of anendoglucanase, a cellobiohydrolase, and a beta-glucosidase.

[44] The process of paragraph 42, wherein the hemicellulase is one ormore (e.g., several) enzymes selected from the group consisting of axylanase, an acetylxylan esterase, a feruloyl esterase, anarabinofuranosidase, a xylosidase, and a glucuronidase.

[45] The process of any of paragraphs 40-44, wherein steps (a) andoptionally (b) are performed simultaneously in a simultaneoussaccharification and fermentation.

[46] The process of any of paragraphs 40-45, wherein the fermentationproduct is an alcohol, an alkane, a cycloalkane, an alkene, an aminoacid, a gas, isoprene, a ketone, an organic acid, or polyketide.

[47] A process of fermenting a cellulosic material, comprising:fermenting the cellulosic material with one or more (e.g., several)fermenting microorganisms, wherein the cellulosic material issaccharified with an enzyme composition in the presence of thepolypeptide having catalase activity of any of paragraphs 1-9.

[48] The process of paragraph 47, wherein the fermenting of thecellulosic material produces a fermentation product.

[49] The process of paragraph 48, further comprising recovering thefermentation product from the fermentation.

[50] The process of any of paragraphs 47-49, wherein the cellulosicmaterial is pretreated before saccharification.

[51] The process of any of paragraphs 47-50, wherein the enzymecomposition comprises one or more (e.g., several) enzymes selected fromthe group consisting of a cellulase, a GH61 polypeptide havingcellulolytic enhancing activity, a hemicellulase, an esterase, anexpansin, a laccase, a ligninolytic enzyme, a pectinase, a peroxidase, aprotease, and a swollenin.

[52] The process of paragraph 51, wherein the cellulase is one or more(e.g., several) enzymes selected from the group consisting of anendoglucanase, a cellobiohydrolase, and a beta-glucosidase.

[53] The process of paragraph 51, wherein the hemicellulase is one ormore (e.g., several) enzymes selected from the group consisting of axylanase, an acetylxylan esterase, a feruloyl esterase, anarabinofuranosidase, a xylosidase, and a glucuronidase.

[54] The process of any of paragraphs 47-53, wherein the fermentationproduct is an alcohol, an alkane, a cycloalkane, an alkene, an aminoacid, a gas, isoprene, a ketone, an organic acid, or polyketide.

[55] A whole broth formulation or cell culture composition comprisingthe polypeptide of any of paragraphs 1-9.

[56] A method for removing hydrogen peroxide, comprising treating amixture to which hydrogen peroxide has been added or generated with thepolypeptide of any of paragraphs 1-9.

[57] A method for generating molecular oxygen, comprising treating amixture to which hydrogen peroxide has been added or generated with thepolypeptide of any of paragraphs 1-9.

[58] A method for removing hydrogen peroxide from textile, comprisingtreating the textile with the polypeptide having catalase activity ofany of paragraphs 1-9.

The invention described and claimed herein is not to be limited in scopeby the specific aspects herein disclosed, since these aspects areintended as illustrations of several aspects of the invention. Anyequivalent aspects are intended to be within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims. In the case ofconflict, the present disclosure including definitions will control.

What is claimed is:
 1. A process for degrading or hydrolyzing acellulosic material, comprising: contacting the cellulosic material witha cellulolytic enzyme composition and an isolated polypeptide to therebydegrade or hydrolyze the cellulosic material, wherein said isolatedpolypeptide has catalase activity and is selected from the groupconsisting of: a) a polypeptide comprising an amino acid sequence havingat least 95% sequence identity to the sequence of amino acids 20 to 741of SEQ ID NO: 8, and b) a fragment of the sequence of amino acids 20 to741 of SEQ ID NO: 8, wherein the fragment has catalase activity.
 2. Theprocess of claim 1, wherein said isolated polypeptide comprises an aminoacid sequence having at least 97% sequence identity to the sequence ofamino acids 20 to 741 of SEQ ID NO:
 8. 3. The process of claim 1,wherein said isolated polypeptide comprises an amino acid sequencehaving at least 98% sequence identity to the sequence of amino acids 20to 741 of SEQ ID NO:
 8. 4. The process of claim 1, wherein said isolatedpolypeptide comprises an amino acid sequence having at least 99%sequence identity to the sequence of amino acids 20 to 741 of SEQ ID NO:8.
 5. The process of claim 1, wherein said isolated polypeptidecomprises the sequence of amino acids 20 to 741 of SEQ ID NO:
 8. 6. Theprocess of claim 1, wherein said isolated polypeptide is encoded by apolynucleotide that hybridizes under high stringency conditions with thefull-length complement of the nucleic acid sequence of nucleotides 58 to2476 of SEQ ID NO: 7, wherein the high stringency conditions are definedas prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200micrograms/ml sheared and denatured salmon sperm DNA, and 50% formamidefor 12 to 24 hours, followed by washing three times each for 15 minutesusing 2×SSC, 0.2% SDS at 65° C.
 7. The process of claim 1, wherein saidisolated polypeptide is encoded by a polynucleotide comprising a nucleicacid sequence having at least 95% sequence identity to the nucleic acidsequence of nucleotides 58 to 2476 of SEQ ID NO:
 7. 8. The process ofclaim 1, wherein said isolated polypeptide is encoded by apolynucleotide comprising a nucleic acid sequence having at least 97%sequence identity to the nucleic acid sequence of nucleotides 58 to 2476of SEQ ID NO:
 7. 9. The process of claim 1, wherein said isolatedpolypeptide is encoded by a polynucleotide comprising a nucleic acidsequence having at least 99% sequence identity to the nucleic acidsequence of nucleotides 58 to 2476 of SEQ ID NO:
 7. 10. The process ofclaim 1, wherein said isolated polypeptide is a variant of the sequenceof amino acids 20 to 741 of SEQ ID NO: 8, wherein the variant ismodified by a substitution, deletion, and/or insertion at one or morepositions in the amino acid sequence of 20 to 741 of SEQ ID NO: 8, andwherein said isolated polypeptide comprises an amino acid sequencehaving at least 95% sequence identity to the sequence of amino acids 20to 741 of SEQ ID NO: 8 and has catalase activity.
 11. A process forproducing a fermentation product, comprising: (a) saccharifying acellulosic material with a cellulolytic enzyme composition and anisolated polypeptide to produce a saccharified cellulosic material,wherein the isolated polypeptide has catalase activity and is selectedfrom the group consisting of: i) a polypeptide comprising an amino acidsequence having at least 95% sequence identity to the sequence of aminoacids 20 to 741 of SEQ ID NO: 8, and ii) a fragment of the sequence ofamino acids 20 to 741 of SEQ ID NO: 8, wherein the fragment has catalaseactivity; (b) fermenting the saccharified cellulosic material with oneor more fermenting microorganisms to produce the fermentation product;and optionally (c) recovering the fermentation product from thefermentation.
 12. The process of claim 11, wherein said isolatedpolypeptide comprises an amino acid sequence having at least 97%sequence identity to the sequence of amino acids 20 to 741 of SEQ ID NO:8.
 13. The process of claim 11, wherein said isolated polypeptidecomprises an amino acid sequence having at least 98% sequence identityto the sequence of amino acids 20 to 741 of SEQ ID NO:
 8. 14. Theprocess of claim 11, wherein said isolated polypeptide comprises anamino acid sequence having at least 99% sequence identity to thesequence of amino acids 20 to 741 of SEQ ID NO:
 8. 15. The process ofclaim 11, wherein said isolated polypeptide comprises the sequence ofamino acids 20 to 741 of SEQ ID NO:
 8. 16. The process of claim 11,wherein said isolated polypeptide is encoded by a polynucleotide thathybridizes under high stringency conditions with the full-lengthcomplement of the nucleic acid sequence of nucleotides 58 to 2476 of SEQID NO: 7, wherein the high stringency conditions are defined asprehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200micrograms/ml sheared and denatured salmon sperm DNA, and 50% formamidefor 12 to 24 hours, followed by washing three times each for 15 minutesusing 2×SSC, 0.2% SDS at 65° C.
 17. The process of claim 11, whereinsaid isolated polypeptide is encoded by a polynucleotide comprising anucleic acid sequence having at least 95% sequence identity to thenucleic acid sequence of nucleotides 58 to 2476 of SEQ ID NO:
 7. 18. Theprocess of claim 11, wherein said isolated polypeptide is encoded by apolynucleotide comprising a nucleic acid sequence having at least 97%sequence identity to the nucleic acid sequence of nucleotides 58 to 2476of SEQ ID NO:
 7. 19. The process of claim 11, wherein said isolatedpolypeptide is encoded by a polynucleotide comprising a nucleic acidsequence having at least 99% sequence identity to the nucleic acidsequence of nucleotides 58 to 2476 of SEQ ID NO:
 7. 20. The process ofclaim 11, wherein said isolated polypeptide is a variant of the sequenceof amino acids 20 to 741 of SEQ ID NO: 8, wherein the variant ismodified by a substitution, deletion, and/or insertion at one or morepositions in the amino act sequence of 20 to 741 of SEQ ID NO: 8, andwherein said isolated polypeptide comprises an amino acid sequencehaving at least 95% sequence identity to the sequence of amino acids 20to 741 of SEQ ID NO: 8 and has catalase activity.